The present invention relates to peptides of one or more portions of the human chorionic gonadotropin xcex2-chain as well as methods for treatment and prevention of diseases, including HIV infection, cancer and wasting syndrome, and methods of promoting hematopoiesis using human chorionic gonadotropin, employing the xcex2-chain of human chorionic gonadotropin, peptides containing a sequence of one or more portions of the xcex2-chain of human chorionic gonadotropin and derivatives and analogues thereof. The invention further relates to fractions of sources and or preparations of human chorionic gonadotropin, such as fractions of human early pregnancy urine, which fractions have anti-HIV activity, anti-cancer activity, anti-wasting activity, and/or pro-hematopoietic activity. The present invention further relates to pharmaceutical compositions for treating and/or preventing HIV infection, cancer, and/or wasting, and/or for promoting hematopoiesis.
The Human Immunodeficiency Virus
The human immunodeficiency virus (HIV) has been implicated as the primary cause of the slowly degenerative immune system disease termed acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F., et al., 1983, Science 220:868-870; Gallo, R., et al., 1984, Science 224:500-503). There are at least two distinct types of HIV: HIV-1 (Barre-Sinoussi, F., et al., 1983, Science 220:868-870; Gallo, R., et al., 1984, Science 224:500-503) and HIV-2 (Clavel, F., et al., 1986, Science 233:343-346; Guyader, M., et al., 1987, Nature 326:662-669). Further, a large amount of genetic heterogeneity exists within populations of each of these types. In humans, HIV replication occurs prominently in CD4+ T lymphocyte populations, and HIV infection leads to depletion of this cell type and eventually to immune incompetence, opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death.
HIV is a member of the lentivirus family of retroviruses (Teich, N., et al., 1984, RNA Tumor Viruses, Weiss, R., et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a single-stranded RNA genome and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439).
The HIV viral particle comprises a viral core, composed in part of capsid proteins, together with the viral RNA genome and those enzymes required for early replicative events. Myristylated gag protein forms an outer shell around the viral core, which is, in turn, surrounded by a lipid membrane envelope derived from the infected cell membrane. The HIV envelope surface glycoproteins are synthesized as a single 160 kilodalton precursor protein which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane glycoprotein and gp120 is an extracellular glycoprotein which remains non-covalently associated with gp41, possibly in a trimeric or multimeric form (Hammarskjold, M., and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280).
HIV is targeted to CD4+ cells because a CD4 cell surface protein (CD4) acts as the cellular receptor for the HIV-1 virus (Dalgleish, A., et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon gp120 binding the cellular CD4 receptor molecules (McDougal, J. S., et al., 1986, Science 231:382-385; Maddon, P. J., et al., 1986, Cell 47:333-348), explaining HIV""s tropism for CD4+ cells, while gp41 anchors the envelope glycoprotein complex in the viral membrane. While these virus:cell interactions are necessary for infection, there is evidence that additional virus:cell interactions are also required.
HIV Treatment
HIV infection is pandemic and HIV-associated diseases represent a major world health problem. Although considerable effort is being put into the design of effective Therapeutics, currently no curative anti-retroviral drugs against AIDS exist. In attempts to develop such drugs, several stages of the HIV life cycle have been considered as targets for therapeutic intervention (Mitsuya, H., et al., 1991, FASEB 1. 5:2369-2381). Many viral targets for intervention with HIV life cycle have been suggested, as the prevailing view is that interference with a host cell protein would have deleterious side effects. For example, virally encoded reverse transcriptase has been one focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2xe2x80x2,3xe2x80x2-dideoxynucleoside analogs such as AZT, ddl, ddC, and d4T have been developed which have been shown to been active against HIV (Mitsuya, H., et al., 1991, Science 249:1533-1544).
The new treatment regimens for HIV-1 show that a combination of anti-HIV compounds, which target reverse transcriptase (RT), such as azidothymidine (AZT), lamivudine (3TC), dideoxyinosine (ddl), dideoxycytidine (ddC) used in combination with an HIV-1 protease inhibitor have a far greater effect (2 to 3 logs reduction) on viral load compared to AZT alone (about 1 log reduction). For example, impressive results have recently been obtained with a combination of AZT, ddl, 3TC and ritonavir (Perelson, A. S., et al., 1996, Science 15:1582-1586). However, it is likely that long-term use of combinations of these chemicals will lead to toxicity, especially to the bone marrow. Long-term cytotoxic therapy may also lead to suppression of CD8+ T cells, which are essential to the control of HIV, via killer cell activity (Blazevic, V., et al., 1995, AIDS Res. Hum. Retroviruses 11:1335-1342) and by the release of suppressive factors, notably the chemokines Rantes, MIP-1xcex1 and MIP-1xcex2 (Cocchi, F., et al., 1995, Science 270:1811-1815). Another major concern in long-term chemical anti-retroviral therapy is the development of HIV mutations with partial or complete resistance (Lange, J. M., 1995, AIDS Res. Hum. Retroviruses 10:S77-82). It is thought that such mutations may be an inevitable consequence of anti-viral therapy. The pattern of disappearance of wild-type virus and appearance of mutant virus due to treatment, combined with coincidental decline in CD4+ T cell numbers strongly suggests that, at least with some compounds, the appearance of viral mutants is a major underlying factor in the failure of AIDS therapy.
Attempts are also being made to develop drugs which can inhibit viral entry into the cell, the earliest stage of HIV infection. Here, the focus has thus far been on CD4, the cell surface receptor for HIV. Recombinant soluble CD4, for example, has been shown to inhibit infection of CD4+ T cells by some HIV-1 strains (Smith, D. H., et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar, E., et al., 1990, Proc. Nati. Acad. Sci. USA 87:6574-6579). In addition, recombinant soluble CD4 clinical trials have produced inconclusive results (Schooley, R., et al., 1990, Ann. Int. Med. 112:247-253; Kahn, J. O., et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan, R., et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
The late stages of HIV replication, which involve crucial virus-specific processing of certain viral encoded proteins, have also been suggested as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs are being developed which inhibit this protease (Erickson, J., 1990, Science 249:527-533).
Recently, chemokines produced by CD8+ T cells have been implicated in suppression of HIV infection (Paul, W. E., 1994, Cell 82:177; Bolognesi, D. P., 1993, Semin. Immunol. 5:203). The chemokines RANTES, MIP-1xcex1 and MIP-1xcex2, which are secreted by CD8+ T cells, were shown to suppress HIV-1 p24 antigen production in cells infected with HIV-1 or HIV-2 isolates in vitro (Cocchi, F, et al., 1995, Science 270:1811-1815). Thus, these and other chemokines may prove useful in therapies for HIV infection. The clinical outcome, however, of all these and other candidate drugs is still in question.
Attention is also being given to the development of vaccines for the treatment of HIV infection. The HIV-1 envelope proteins (gp160, gp120, gp41) have been shown to be the major antigens for anti-HIV antibodies present in AIDS patients (Barin et al., 1985, Science 228:1094-1096). Thus far, therefore, these proteins appear to be the most promising candidates to act as antigens for anti-HIV vaccine development. Several groups have begun to use various portions of gp160, gp120, and/or gp41 as immunogenic targets for the host immune system. See for example, Ivanoff, L., et al., U.S. Pat. No. 5,141,867; Saith, G., et al., WO92/22,654; Shafferman, A., WO91/09,872; Formoso, C., et al., WO90/07,119. Vaccines directed against HIV proteins are problematic in that the virus mutates rapidly rendering many of these vaccines ineffective. Thus, although a great deal of effort is being directed to the design and testing of anti-retroviral drugs, effective, non-toxic treatments are still needed.
Hematopoietic Cell Production
The morphologically recognizable and functionally capable cells circulating in blood include erythrocytes, neutrophilic, eosinophilic, and basophilic granulocytes, B-, T-, non B-, non T-lymphocytes, and platelets. These mature hematopoietic cells derive from and are replaced, on demand, by morphologically recognizable dividing precursor cells for the respective lineages such as erythroblasts for the erythrocyte series, myeloblasts, promyelocytes and myelocytes for the granulocyte series, and megakaryocytes for the platelets. The precursor cells derive from more primitive cells that can simplistically be divided into two major subgroups: stem cells and progenitor cells (for review, see Broxmeyer, H. E., 1983, xe2x80x9cColony Assays of Hematopoietic Progenitor Cells and Correlations to Clinical Situations,xe2x80x9d CRC Critical Reviews in Oncology/Hematology 1:227-257).
The definitions of stem and progenitor cells are operational and depend on functional, rather than on morphological, criteria. Stem cells have extensive self-renewal or self-maintenance capacity (Lajtha, L. G., 1979, Differentiation 14:23), a necessity since absence or depletion of these cells could result in the complete depletion of one or more cell lineages, events that would lead within a short time to disease and death. Some of the stem cells differentiate upon need, but some stem cells or their daughter cells produce other stem cells to maintain the pool of these cells. Thus, in addition to maintaining their own kind, pluripotential stem cells are capable of differentiation into several sub-lines of progenitor cells with more limited self-renewal capacity or no self-renewal capacity. These progenitor cells ultimately give rise to the morphologically recognizable precursor cells. The progenitor cells are capable of proliferating and differentiating along one, or more than one, of the myeloid differentiation pathways (Lajtha, L. G. (Rapporteur), 1979, Blood Cells 5:447).
A variety of infectious agents, genetic abnormalities and environmental factors can cause a deficiency in one or more hematopoietic cell types. For example, hematological abnormalities have been observed in HIV-1 infected individuals (the human immunodeficiency virus (HIV) has been implicated as the primary cause of the slowly degenerative immune system disease termed acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F., et al., 1983, Science 220:868-870; Gallo, R., et al., 1984, Science 224:500-503)), particularly in the late stages of disease (Lunardi-Iskandar, Y. et al., 1989, J. Clin. Invest 83:610-615). These abnormalities include a reduction in CD4+ T cells as well as cytopenias of one or more hematopoietic lineages, often associated with bone marrow morphologic abnormalities and deficient progenitor cell growth (Lunardi-Iskandar, Y. et al., 1989, J. Clin. Invest 83:610-615; Louache, F. et al., 1992, Blood 180:2991-2999). Idiopathic thrombocytopenic purpura (ITP), characterized by significant reduction in platelet numbers, often afflicts subjects infected with HIV (Ballem, P. J. et al., 1992, N. Engl. J. Med. 327:1779). The destruction of platelets in sufferers of ITP appears to be mediated by platelet associated autoantibodies (Berchtold, P. and Wenger, M., 1993, Blood 81:1246; Ballem, P. J. et al., 1987, J. Clin. Invest. 80:33). Thus, because management of ITP generally involves immunosuppression, treatment of ITP in HIV infected patients is complicated as administration of immunosuppressive drugs is extremely detrimental in HIV infection.
Additionally, chemotherapy and radiation therapy used in the treatment of cancer and certain immunological disorders can cause pancytopenias or combinations of anemia, neutropenia and thrombocytopenia. Thus, the increase or replacement of hematopoietic cells is often crucial to the success of such treatments. (For a general discussion of hematological disorders and their causes, see, e.g., xe2x80x9cHematologyxe2x80x9d in Scientific American Medicine, E. Rubenstein and D. Federman, eds., Volume 2, chapter 5, Scientific American, New York (1996)).
Furthermore, aplastic anemia presents a serious clinical condition as the overall mortality of all patients with aplastic anemias, in the absence of stem cell therapy, is high. Approximately 60-75% of individuals suffering from the disorder die within 12 months, in the absence of new stem cells. The overall incidence of these diseases is approximately 25 new cases per million persons per year. Although it is extremely unlikely that a single pathogenic mechanism accounts for all aplastic anemias, it is clear that provision of new hematopoietic stem cells is usually sufficient to allow permanent recovery, since transplantation of patients with aplastic anemia with bone marrow obtained from identical twins (i.e., syngeneic) (Pillow, R. P., et al., 1966, N. Engl. J. Med. 275:94-97) or from HLA-identical siblings (i.e., allogeneic) (Thomas, E. D., et al., Feb. 5, 1972, The Lancet, pp. 284-289) can fully correct the disease. However, some patients with aplastic anemia reject the transplanted marrow. This complication is particularly common among patients who have been immunologically sensitized as a result of multiple therapeutic blood, transfusions.
The current therapy available for many hematological disorders as well as the destruction of the endogenous hematopoietic cells caused by chemotherapy or radiotherapy is bone marrow transplantation. However, use of bone marrow transplantation is severely restricted since it is extremely rare to have perfectly matched (genetically identical) donors, except in cases where an identical twin is available or where bone marrow cells of a patient in remission are stored in a viable frozen state. Except in such autologous cases, there is an inevitable genetic mismatch of some degree, which entails serious and sometimes lethal complications. These complications are two-fold. First, the patient is usually immunologically incapacitated by drugs beforehand, in order to avoid immune rejection of the foreign bone marrow cells (host versus graft reaction). Second, when and if the donated bone marrow cells become established, they can attack the patient (graft versus host disease), who is recognized as foreign. Even with closely matched family donors, these complications of partial mismatching are the cause of substantial mortality and morbidity directly due to bone marrow transplantation from a genetically different individual.
Peripheral blood has also been investigated as a source of stem cells for hematopoietic reconstitution (Nothdurtt, W., et al., 1977, Scand. J. Haematol. 19:470-481; Sarpel, S. C., et al., 1979, Exp. Hematol. 7:113-120; Ragharachar, A., et al., 1983, J. Cell. Biochem. Suppl. 7A:78; Juttner, C. A., et al., 1985, Brit. J. Haematol. 61:739-745; Abrams, R. A., et al., 1983, J. Cell. Biochem. Suppl. 7A:53; Prummer, O., et al., 1985, Exp. Hematol. 13:891-898). In some studies, promising results have been obtained for patients with various leukemias (Reiffers, J., et al., 1986, Exp. Hematol. 14:312-315; Goldman, J. M., et al., 1980, Br. J. Haematol. 45:223-231; Tilly, H., et al., July 19, 1986, The Lancet, pp. 154-155; see also To, L. B. and Juttner, C. A., 1987, Brit. J. Haematol. 66: 285-288, and references cited therein); and with lymphoma (Korbling, M., et al., 1986, Blood 67:529-532). Other studies using peripheral blood, however, have failed to effect reconstitution (Hershko, C., et al., 1979, The Lancet 1:945-947; Ochs, H. D., et al., 1981, Pediatr. Res. 15:601). Studies have also investigated the use of fetal liver cell transplantation (Cain, G. R., et al., 1986, Transplantation 41:32-25; Ochs, H. D., et al., 1981, Pediatr. Res. 15:601; Paige, C. J., et al., 1981, J. Exp. Med. 153:154-165; Touraine, J. L., 1980, Excerpta Med. 514:277; Touraine, J. L., 1983, Birth Defects 19:139; see also Good, R. A., et al., 1983, Cellular Immunol. 82:44-45 and references cited therein) or neonatal spleen cell transplantation (Yunis, E. J., et al., 1974, Proc. Natl. Acad. Sci. U.S.A. 72:4100) as stem cell sources for hematopoietic reconstitution. Cells of neonatal thymus have also been transplanted in immune reconstitution experiments (Vickery, A. C., et al., 1983, J. Parasitol. 69(3):478-485; Hirokawa, K., et al., 1982, Clin. Immunol. Immunopathol. 22:297-304).
Clearly, there is a tremendous need for methods of expanding blood cells in vitro or therapies which increase the production of hematopoietic cells in vivo.
Wasting Syndromes
Wasting syndrome is a serious clinical problem characterized by a decrease in body mass of more than 10% from baseline body weight and a disproportionate loss of body mass with respect to body fat (Weinroth et al., 1995, Infectious Agents and Disease 4:76-94; Kotler and Grunfeld, 1995, AIDS Clin. Rev. 96:229-275). Thus, wasting is distinguished from starvation in which higher levels of body fat than body cell mass are depleted (Kotler et al., 1985, Am J. Clin. Nutr. 42:1255-1265; Cahill, 1970, N. Engl. J. Med. 282:668-675). Wasting is associated with a variety of conditions, including HIV infection (human immunodeficiency virus (HIV) has been implicated in acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F., et al., 1983, Science 220:868-870; Gallo, R., et al., 1984, Science 224:500-503)), other infectious diseases, sepsis, cancer, chronic cardiovascular disease and diarrhea (Kotler et al., 1989, Am. J. Clin. Nutr. 50:444-447; Heymsfield et al., 1982, Am. J. Clin. Nutr. 36:680-690). Importantly, wasting is a significant factor in the mortality of patients suffering from infections or cancer. In fact, body cell mass depletion has a linear relationship to time of survival in AIDS patients (Kotler et al., 1989, Am. J. Clin. Nutr. 50:444-447).
The cause of wasting syndrome in AIDS and other conditions is unclear and is most likely multifactorial. Metabolic abnormalities, irregular levels of hormones and cytokines, and malabsorption have all been implicated in wasting syndrome. Not all AIDS patients suffer from wasting, suggesting that the cause of the wasting is not HIV itself. Most cases of HIV associated wasting syndrome are apparently caused by complications of AIDS, such as secondary infections and gastrointestinal disease (Kotler and Grunfeld, 1995, AIDS Clin. Rev. 96:229-275).
Current and potential therapies for wasting syndromes include nutritional support, appetite enhancers such as dronabinol and megestrol acetate, anabolic therapies, such as growth hormone, and cytokine inhibitors. However, mixed results have been obtained with nutritional support and appetite enhancers in that patients tended to gain only fat and not overall body mass. Administration of growth hormone, and cytokine inhibitors are still being tested and may pose a risk of side effects (Kotler and Grunfeld, 1995, AIDS Clin. Rev. 96:229-275; Weinroth et al., 1995, Infectious Agents and Disease 4:76-94).
Thus, treatment of wasting is critical to the survival and well-being of patients suffering from serious diseases such as cancer and AIDS; thus, there is a need for safe and effective therapies for wasting syndrome associated with cancer, AIDS and other infectious diseases.
Cancer
A neoplasm, or tumor, is a neoplastic mass resulting from abnormal uncontrolled cell growth, which may cause swelling on the body surface, and which can be benign or malignant. Benign tumors generally remain localized. Malignant tumors are collectively termed cancers. The term xe2x80x9cmalignantxe2x80x9d generally means that the tumor can invade and destroy neighboring body structures and spread to distant sites to cause death (for review, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-122). Treatment options, such as surgery, chemotherapy and radiation treatment, are either ineffective or present serious side effects. Thus, there is a need for development of new drugs for the treatment of cancer.
Kaposi""s Sarcoma (KS) is a rare type of cancer, the incidence of which is greatly increased in HIV infected people (Lunardi-Iskandar, Y., et al., 1995, Nature 375:64-68; Friedman-Kien, A. E., et al., 1981, J. Am. Acad. Dermatol. 5:468-473). The tumors appear to be comprised of hyperplastic cells derived from vascular endothelial cells (Nakamura, S., et al., 1988, Science 242:426-430; Ensoli, B., et al, 1989, Science 243:223-226; Salahuddin, S. Z., et al., 1988, Science 242:430-433; Masood, R., et al., 1994, AIDS Res. Hum. Retroviruses 10:969-976; Lunardi-Iskandar, Y., et al., 1995, JNCI 88:450-454). In some cases, neoplastic cells with chromosomal abnormalities are also present in the tumors (Lunardi-Iskandar, Y., et al., 1995, JNCI 87:974-981; Delli-Bovi, P., et al., 1986, Cancer Res. 46:6333-6338; Siegal, B., et al., 1990, Cancer 65:492-498; Yunis, J. J., 1983, Science 221:227-236; Popescu, N. C., et al., 1995, JNCI 88:450-454). Therapies for KS include radiotherapy, xcex1-interferon and systemic chemotherapy (Chak, L. Y., et al., 1988, J. Clin. Oncol. 6:863-7; Evans, L. M., et al., 1991, J. Immunother. 10:39-50; Kovas, J., et al., 1990, Ann. Intern. Med. 112:812-21; Gelmann, E. D., et al., 1987, Am. J. Med. 82:456-62; Gill, P. S., et al., 1991, Am. J. Med. 90:427-33; Gill, P. S., et al., 1990; Am. J. Clin. Oncol. 13:315-9; Gill, P. S., et al., 1994, AIDS 8:1695-9). However, hematological and non-hematological toxicities limit the prolonged use of chemotherapy and xcex1-interferon in conjunction with anti-retroviral agents commonly used in the treatment of AIDS (Kovas, J., et al., 1990, Ann. Intern. Med. 112:812-21; Gill, P. S., et al., 1991, Am. J. Med. 90:427-33; Gill, P. S., et al., 1994, AIDS 8:1695-9). Thus, new drugs, preferably drugs compatible with AIDS therapeutics, are needed for the treatment of KS.
Human Chorionic Gonadotropin
Human chorionic gonadotropin (hCG), which is required for the maintenance of pregnancy, is a member of the glycoprotein hormone family. The glycoprotein hormones, which also include follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH), consist of two sub-units, xcex1 and xcex2. These subunits are non-covalently linked to form a heterodimer, and heterodimer formation has been shown to be required for receptor binding. Within a particular species, the xcex1-subunits are identical among the glycoprotein hormones while the xcex2-subunits differ and determine the receptor binding specificity of the particular hormone (Kornyei, J. L., et al., 1993, Biol. Reprod. 49:1149). The xcex2-subunits of the glycoprotein hormones exhibit a high degree of sequence similarity within the N-terminal 114 amino acids. LH is the most similar to hCG with 85% sequence homology within the first 114 amino acids, and both proteins bind the same receptor. hCG, however, contains a C-terminal extension not present in the other glycoprotein xcex2-chains (Lapthorn, A. J., et al., 1994, Science 369:455-461).
From the three dimensional crystal structure of hCG, it has been determined that hCG, like the growth factors nerve growth factor (NGF), transforming growth factor-xcex2 (TGF-xcex2) and platelet-derived growth factor-xcex2 (PDGF-xcex2), is a cysteine-knot glycoprotein. Proteins containing such a cysteine-knot motif have at least three disulfide bridges, two of which join adjacent anti-parallel strands of the peptide, thus, forming a ring, and one of which joins the peptide chain through the ring. Particular structures in the hCG xcex2-chain include the determinant loop sequence (xcex293-100) which has been implicated in subunit association and the longest inter-cysteine loop (xcex238-57) which may play a role in receptor binding. Residues 47-53 appear to be exposed at the surface of this inter-cysteine loop (Lapthorn et al., 1994, Nature 369:455-461).
Previously, purified preparations of heterodimeric hCG have been shown to reduce the reverse transcriptase activity in HIV-1 infected lymphocytes and monocytes in culture (Bourinbaiar, A. S., and Nagorny, R., 1992, FEMS Microbiology Letters 96:27-30) and to prevent transmission of HIV from lymphocytes to trophoblasts in vitro (Bourinbaiar, A. S., and Nagorny, R., 1992, FEBS Letters 309:82-84). Additionally, the xcex2-subunit of hCG (xcex2-hCG) has been demonstrated to reduce HIV production in lymphocytes at doses from 100 pg/ml to 100 xcexcg/ml and in monocytes at doses up to approximately 10 xcexcg/ml, with higher doses actually increasing the level of viral production in monocytes (Bourinbaiar, A. S., and Lee-Huang, S., 1995, Immunology Letters 44:13-17). However, none of these reports disclose the potential efficacy of xcex2-hCG peptides in HIV inhibition in vitro or of hCG or any portion or derivative thereof in HIV treatment or prevention in vivo.
Furthermore, doses of hCG below those necessary to induce a humoral immune response have been proposed for treatment of HIV infection based on observations of therapeutic effects of such doses on cats and cows infected with feline leukemia and bovine leukemia viruses respectively (U.S. Pat. No. 4,880,626). This patent suggested use of the hCG dimer at very low doses (approximately 2 I.U. per treatment).
Lunardi-Iskandar et al. (1995, Nature 375:64-68 and PCT Application WO96/04008) reported that hCG, xcex2-hCG, as well as a xcex2-hCG carboxy-terminal peptides of amino acids 109-145 (SEQ ID NO:25) and 109-119 (SEQ ID NO:7) are efficacious in the treatment of Kaposi""s Sarcoma. However, neither reference discloses or even suggests that hCG, xcex2-hCG or xcex2-hCG peptides of amino acids 109-145 or 109-119 (SEQ ID NOS:7 and 25, respectively) have any viral anti-activity or that other xcex2-hCG peptides have any therapeutic activity.
Finally, Harris (1995, The Lancet 346:118-119) reported that treatment with hCG improved T cell counts and physical symptoms in certain HIV infected subjects.
The present invention fulfills a need for safe and effective therapies for HIV, wasting syndrome associated with AIDS, other infectious diseases and cancer, as well as methods for promoting hematopoiesis.
Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention.
The present inventors have found that hCG preparations, xcex2-hCG preparations, certain peptides of xcex2-hCG, certain combinations of xcex2-hCG peptides linked via their N-termini and C-termini by peptide bond(s), and certain gel filtration chromatography fractions of commercial hCG preparations and human early (i.e. first trimester) pregnancy urine exhibit anti-viral activities, including anti-HIV activities. In particular, hCG and xcex2-hCG preparations and certain gel filtration chromatography fractions of commercial hCG preparations and of human early pregnancy urine, as described by way of example hereinbelow, and specific peptides thereof inhibit HIV-1 replication in vitro, inhibit HIV-1 gene-expression in HIV-1 transgenic mice, reduce plasma virus levels in SIV infected monkeys and in AIDS patients, and increase CD4+ T cells in HIV transgenic mice, SIV infected monkeys and AIDS patients. The present inventors have further found that the subjects tolerated treatment with hCG and xcex2-hCG preparations very well and that the virus did not become resistant to treatment after exposure to hCG or xcex2-hCG. The present invention fills a tremendous need for a non-toxic, long-term treatment of HIV infection and its sequelae, ARC and AIDS.
The present invention relates to proteins having a sequence of one or more portions of the xcex2-chain of hCG (xcex2-hCG), particularly proteins having the sequence of amino acid numbers 41-54, 45-54, 47-53, 45-57 and 109-119 (SEQ ID NOS:3-7, respectively). The present invention also relates to proteins comprising or, alternatively, consisting of, the sequence of two or more portions of xcex2-hCG, e.g., wherein said portions are linked via their N-termini and C-termini by peptide bond(s), particularly proteins having the sequence of amino acid numbers 45-57 (SEQ ID NO:6) linked via a peptide bond at the C-terminus to the N-terminus of a peptide of amino acid numbers 109-119 (SEQ ID NO:7) or linked at the N-terminus to the C-terminus of a peptide of amino acid numbers 110-119 (SEQ ID NO:27); or a peptide of amino acid numbers 47-57 (SEQ ID NO:28) linked by a peptide bond at the C-terminus to the N-terminus of a peptide of amino acid numbers 108-119 (SEQ ID NO:29) of xcex2-hCG as depicted in FIG. 8 (a portion of SEQ ID NO:2), i.e. the peptides denoted 45-57::109-119, 110-119::45-57, or 47-57::108-119 (SEQ ID NOS:30-32, respectively). The present invention also relates to certain fractions (i.e. components of a source of hCG or xcex2-hCG isolated away from other components in the source of hCG or xcex2-hCG by a separation technique known in the art) of any source of hCG or xcex2-hCG, such as commercial hCG preparations and human (preferably early, i.e., first trimester) pregnancy urine, which fractions have anti-HIV and/or anti-Kaposi""s Sarcoma activity.
The present invention further relates to therapeutic methods and compositions for treatment and prevention of diseases and disorders associated with HIV infection based on hCG and xcex2-hCG preparations, therapeutically and prophylactically effective fractions of a source of hCG or xcex2-hCG (preferably a source of native hCG or xcex2-hCG, i.e. a source of naturally occurring hCG or xcex2-hCG, not recombinantly produced hCG or xcex2-hCG) and therapeutically and prophylactically effective proteins containing a sequence of one or more portions (i.e., a fusion protein comprising more than one xcex2-hCG peptide sequence either as non-contiguous or contiguous sequences, e.g., having an amino acid sequence of one xcex2-hCG peptide linked via a peptide bond to another xcex2-hCG peptide) of xcex2-hCG, and related derivatives and analogs. The invention provides for treatment and prevention of HIV infection by administration of a therapeutic compound of the invention. The therapeutic compounds of the invention include: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of hCG or xcex2-hCG, therapeutically and prophylactically effective peptides having a sequence of a one or more portions of xcex2-hCG, modified derivatives of hCG, xcex2-hCG and xcex2-hCG peptides, and nucleic acids encoding xcex2-hCG and therapeutically and prophylactically effective peptides having a sequence of one or more portions of xcex2-hCG, and derivatives and analogs of the foregoing. The invention also provides in vitro and in vivo assays for assessing the efficacy of therapeutics of the invention for treatment or prevention of HIV. The invention also provides pharmaceutical compositions and methods of administration of therapeutics of the invention for treatment or prevention of HIV infection.
The present invention further relates to therapeutic methods and compositions having anti-wasting activity for treatment and prevention of wasting syndromes based on hCG and xcex2-hCG preparations, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG and therapeutically and prophylactically effective proteins containing a sequence of a portion or portions (i.e., a fusion protein comprising more than one xcex2-hCG peptide sequence, e.g., having an amino acid sequence of one xcex2-hCG peptide linked via a peptide bond to another xcex2-hCG peptide) of xcex2-hCG, and related derivatives and analogs. The invention provides for treatment and prevention of wasting syndromes by administration of a therapeutic compound of the invention. The therapeutic compounds of the invention include: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of hCG or xcex2-hCG (preferably a source of native hCG or native xcex2-hCG, i.e. a source of naturally occurring hCG or xcex2-hCG and not recombinantly produced hCG or xcex2-hCG), therapeutically and prophylactically effective peptides having a sequence of a portion or portions of xcex2-hCG (i.e. a fusion protein comprising more than one xcex2-hCG peptide sequence either as non-contiguous or contiguous sequences, e.g. having an amino acid sequence of one xcex2-hCG peptide linked via a peptide bond to another xcex2-hCG peptide), modified derivatives of hCG, xcex2-hCG and xcex2-hCG peptides, and nucleic acids encoding xcex2-hCG and therapeutically and prophylactically effective peptides having a sequence of a portion or portions of xcex2-hCG, and derivatives and analogs of the foregoing.
The present invention also relates to the use of certain fractions (i.e. components of a source of hCG or xcex2-hCG isolated away from other components in the source of hCG or xcex2-hCG by a separation technique known in the art) of any source of hCG or xcex2-hCG, such as commercial hCG preparations and human (preferably early, i.e., first trimester) pregnancy urine, which fractions have anti-HIV, anti-cancer activity (such as anti-Keposi""s Sarcoma activity), anti-wasting activity and/or pro-hematopoeitic activity.
The invention also provides in vitro and in vivo assays for assessing the efficacy of therapeutics of the invention for treatment or prevention of HIV infection, cancer, and/or wasting syndromes.
The invention also provides pharmaceutical compositions and methods of administration of Therapeutics of the invention for treatment.
The present invention also relates to therapeutic methods and compositions for treatment and prevention of diseases and disorders in which an increase in one or more types of hematopoietic cells is desirable. The therapeutic compounds of the invention are hCG and xcex2-hCG preparations, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG and therapeutically and prophylactically effective proteins containing a sequence of one or more portions (i.e., a fusion protein comprising more than one xcex2-hCG peptide sequence either as non-contigous or contiguous sequences, e.g., having an amino acid sequence of one xcex2-hCG peptide linked via a peptide bond to another xcex2-hCG peptide) of xcex2-hCG, and related derivatives and analogs. The present invention also relates to use of certain fractions (i.e. components of a source of hCG or xcex2-hCG (preferably native hCG or xcex2-hCG, i.e. not recombinantly produced) isolated away from other components in the source of hCG or xcex2-hCG by a separation technique known in the art) of any source of hCG or xcex2-hCG, such as commercial hCG preparations and human (preferably early, i.e., first trimester) pregnancy urine, which fractions have anti-HIV, anti-cancer activity (such as anti-Kaposi""s Sarcoma activity) anti-wasting activity and/or pro-hematopoietic activity.
The invention provides for treatment and prevention of diseases and disorders (e.g., involving hematopoietic cell deficiencies) by administration either of a therapeutic compound of the invention or of hematopoietic cells, the numbers of which have been increased in vitro by contact with a therapeutic compound of the invention. The invention thus also provides in vitro methods of expanding hematopoietic cells. The therapeutic compounds of the invention include: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG, therapeutically and prophylactically effective peptides having a sequence of one or more portions of xcex2-hCG, modified derivatives of hCG, xcex2-hCG and xcex2-hCG peptides, and nucleic acids encoding xcex2-hCG and therapeutically and prophylactically effective peptides having a sequence of one or more portions of xcex2-hCG, and derivatives and analogs of the foregoing.
In a specific embodiment, gene therapy methods are provided using hCG and xcex2-hCG preparations, therapeutically and prophylactically effective fractions of a source of hCG or xcex2-hCG and therapeutically and prophylactically effective proteins containing a sequence of one or more portions of xcex2-hCG, and related derivatives and analogs to induce proliferation of hematopoietic progenitor or stem cells into which cells a nucleic acid of interest is introduced either before or after proliferation. The proliferation induced by the methods of the invention can be with or without concomitant hematopoietic cell differentiation, and, in a preferred embodiment, is proliferation followed by differentiation of the cells.
The present invention further relates to therapeutic methods and compositions for treatment and prevention of cancers based on hCG and xcex2-hCG preparations, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG and therapeutically and prophylactically effective proteins containing a sequence of one or more portions (i.e., a fusion protein comprising more than one xcex2-hCG peptide sequence either as non-contiguous or contiguous sequences, e.g., having an amino acid sequence of one xcex2-hCG peptide linked via a peptide bond to another xcex2-hCG peptide) of xcex2-hCG, and related derivatives and analogs. The invention provides for treatment and prevention of cancers by administration of a therapeutic compound of the invention. The therapeutic compounds of the invention include: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG, therapeutically and prophylactically effective peptides having a sequence of one or more portions of xcex2-hCG, modified derivatives of hCG, xcex2-hCG and xcex2-hCG peptides, and nucleic acids encoding xcex2-hCG and therapeutically and prophylactically effective peptides having a sequence of one or more portions of xcex2-hCG, and derivatives and analogs of the foregoing.
The present invention also relates to certain fractions (i.e. components of a source of hCG or xcex2-hCG isolated away from other components in the source of hCG or xcex2-hCG by any separation technique known in the art) of any source of hCG or xcex2-hCG, such as commercial hCG preparations and human (preferably early, i.e., first trimester) pregnancy urine, which fractions have anti-HIV and or anti-Kaposi""s Sarcoma activity. The invention also provides in vitro and in vivo assays for assessing the efficacy of therapeutics of the invention for treatment or prevention of cancers.
The invention also provides pharmaceutical compositions and methods of administration of Therapeutics of the invention for treatment.
The invention also provides methods of administration and pharmaceutical compositions containing a Therapeutic of the invention.
Definitions
As used herein, the following terms shall have the meaning indicated.
AIDS=Acquired Immune Deficiency Syndrome
ARC=AIDS-Related Complex
BFU-E=burst forming unit-erythroid. A hematopoietic progenitor cell which is capable of producing a colony of erythroid progeny cells in semi-solid medium.
CFU=colony forming unit. A cell which is capable of producing a colony of progeny cells in semi-solid medium.
CFU-GEMM=colony forming unit-granulocyte, erythrocyte, monocyte/macrophage, megakaryocyte. A multipotential hematopoietic progenitor cell which is capable of producing a colony composed of granulocyte, erythrocyte, monocyte/macrophage, and megakaryocyte progeny in semi-solid medium.
CFU-GM=colony forming unit-granulocyte, macrophage. A hematopoietic progenitor cell which is capable of producing a colony composed of granulocyte and macrophage progeny in semi-solid medium.
CFU-MK=colony forming unit-megakaryocyte. A hematopoietic progenitor cell which is capable of producing a colony composed of megakaryocyte progeny in semi-solid medium.
CFU-S=colony forming unit-spleen. A multipotential stem cell with self-renewal capacity, which, upon inoculation into a lethally irradiated mouse, is capable of producing a colony (module) on the spleen. CFU-S is not a marrow-repopulating cell; it is a less primitive stem cell which does not provide long-term engraftment in an animal.
CSF=colony stimulating factor
Epo=erythropoietin
FBS=fetal bovine serum. Also known as fetal calf serum.
G-CSF=granulocyte colony stimulating factor
GM-CSF=granulocyte-macrophage colony stimulating factor
HCG=Human Chorionic Gonadotropin
KS=Kaposi""s Sarcoma
OI=Opportunistic Infection
ITP=Idiopathic thrombocytopenic purpura (a severe platelet deficiency)
PB=peripheral blood
PBMC=Peripheral Blood Mononuclear Cell
FIGS. 1A-E. Effects of an hCG preparation, APL(trademark) (Wyeth-Ayerst), xcex2-hCG peptides and certain fractions of hCG APL(trademark) and early pregnancy urine on weight and HIV-1 gene expression in HIV-1 transgenic mice. (A) and (C) Weight change in grams in individual HIV-1 transgenic mice after treatment from day 1 to day 10 postpartum is represented as a bar graph with open bars representing the weight at day 1 and solid bars representing the weight at day 10. (B), (D) and (E) Suppression of HIV-1 gene expression in transgenic mice. The bar graph presents the level of expression in pixels, as determined by chemiluminescence assay of the HIV genes env, tat, rev, and nef in the individual HIV transgenic mice. For (B) and (D), the black bars represent tat expression, the striped bars represent rev expression, the lightly stippled bars represent env expression, the open bars represent nef expression. For (E), the striped bars represent env expression, the solid bars represent rev expression, and the open bars represent nef expression. In (A) and (B), bars 1-3 represent untreated control transgenic mice; bars 4-6 represent mice whose mothers received (subjects were administered through the mothers"" milk) 200 xcexcg circularized xcex2-hCG peptide 44-57 (with cysteine substituted at position 44; SEQ ID NO:26) per day; bars 7-9 represent mice whose mothers received 300 IU per day hCG-APL(trademark); and bars 9-11 represent mice whose mothers received 200 xcexcg per day of the fused xcex2-hCG peptide 45-57::109-119 (SEQ ID NO:30). In (C) and (D), the bars labeled xe2x80x9cuntreatedxe2x80x9d represent mice receiving only PBS; and the bars labeled xe2x80x9ctreatedxe2x80x9d or xe2x80x9ctreated with HAFcxe2x80x9d represent mice administered 300 IU per day of hCG APL(trademark). In (E), the set of bars labeled xe2x80x9c1xe2x80x9d represents mice treated with PBS alone; xe2x80x9c2xe2x80x9d represents mice treated with 100 xcexcg xcex2-hCG core peptide per day; xe2x80x9c3xe2x80x9d represents mice treated with 100 xcexcg per day oc-hCG; xe2x80x9c4xe2x80x9d represents mice treated with 200 xcexcl per day of fraction 61 of the hCG APL(trademark) fractionation; and xe2x80x9c5xe2x80x9d represents mice treated with 200 xcexcl per day of fraction 65 of the early pregnancy urine fractionation.
FIGS. 2A-D. Effects of an hCG preparation on indicators of SIV infection in SIV-infected macaques. SIV was given intravenously at a dose of 104.5 TCID50 per ml. (A) SIV titer was monitored over time in months by quantifying the p27 gag protein (Organon Teknika assay) as nanograms (ng) of p27/ml of plasma from the plasma of the SIV infected macaques. Treated SIV-infected macaques (indicated as Rx) were given hCG APL(trademark), 3000 IU, 2xc3x97weekly. Plasma levels of p27 gag in these treated monkeys are indicated on the graph by lines with diamonds, number (#) signs or filled circles. Results with the untreated SIV-infected macaques (indicated UnRx) are indicated by the lines with either stars or triangles. (B) CD4+ T cell levels were determined in cells/mm3 in SIV-infected macaques either treated with hCG or untreated over time in months. Results from the SIV-infected monkeys treated with hCG (APL(trademark)) (Rx) are indicated by lines with diamonds, number (#) signs or filled circles, while results with the untreated monkeys (UnRx) are indicated by lines with stars or triangles. (C) Change in weight in kilograms (kg) was monitored in treated and untreated SIV-infected monkeys over time in months. Weight changes in the SIV-infected monkeys treated with hCG (APL(trademark)) (Rx) are indicated by lines with diamonds, # signs or filled circles, while results in the untreated monkeys (UnRx) are indicated by lines with stars or triangles. (D) Levels of CD4+ T cells were monitored in normal uninfected monkeys either treated with hCG (APL(trademark)) or untreated over time in months. CD4+ T cell levels in the untreated monkeys are indicated by lines with sun-like figures or squares, and the results in the treated monkeys are indicated by lines with pentagonal figures or with filled inverted triangles.
FIGS. 3A-J. Effects of administration of hCG preparations on HIV-1 viral load and CD4+ T cell levels in individual patients in the clinical study described herein. Figures A and B are data from patient PHOJ, C and D from patient PG1, E and F from patient PG3, G and H from patient PHVE, and I and J from patient PG17. In panels A, C, E, G and I, viral load and CD4+ T Cell counts are plotted over time (in months). Viral load (measured by RT-PCR in panels A and G and by the Roche Amplicor test in panels C, E and I) is plotted as the logarithm of the viral load (represented by line with xe2x80x9cXxe2x80x9d data points). The CD4+ T Cell levels are plotted as CD4+ T Cells/ml (represented by line with triangle data points). Panels B, D, F, H, and J plot the dosage of hCG in IU (xc3x971000) per week over time in months, with the timing of other therapies indicated above the graph with a thick arrow.
FIGS. 4A-D. Effects of hCG preparations and peptides on HIV replication in vitro. (A and B) These graphs depict the concentration dependence of inhibition of HIV-1 IIIB infection of CD4+ T cells or total PBMCs from peripheral blood of normal donors (infection is expressed in nanograms (ng) of p24/ml plasma) as a function of nmol per ml xcex1-hCG subunit, xcex2-hCG peptide or xcex1-hCG peptide over a concentration of 0.05 to 50 nmol/ml. Graphs present data on infection of (A) CD4+ T cells infected by HIV-1 IIIB and (B) PBMCs infected with HIV-1 IIIB. In both graphs, results with xcex1-hCG subunit are represented by lines with diamonds, results with the xcex2-hCG peptide 6-16 by lines with squares, results with the xcex2-hCG peptide 109-119 by lines with stars, results with the circularized xcex2-hCG peptide 44-57 (with cysteine substituted for the amino acid at position 44) (SEQ ID NO:26) by lines with triangles, and results with the xcex1-hCG peptide 88-92 by lines with inverted triangles. (C and D) These graphs depict data on the effect of xcex1-hCG, hCG peptides and commercial hCG preparations on the infection of primary macrophages by HIV-1 Ba-L. (C) Effect of different concentrations of hCG a subunits and various hCG peptides (0.05 to 50 nmol/ml) on infection in peripheral blood macrophages from a normal donor infected with HIV-1 Ba-L. The results are averages of triplicate samples with less than 15% variation. Results with xcex1-hCG subunit are represented by lines with diamonds, results with the xcex2-hCG peptide 6-16 by lines with squares, results with the xcex2-hCG peptide 109-119 (SEQ ID NO:7) by lines with stars, results with the circularized xcex2-hCG peptide 44-57 (with cysteine substituted for the amino acid at position 44) (SEQ ID NO:26) by lines with triangles, and results with the xcex1-hCG peptide 88-92 by lines with inverted triangles. (D) A comparison of the inhibition of HIV Ba-L infection of macrophages by different commercial native hCG preparations (APL(trademark), Wyeth Ayerst; STERIS(trademark), Steris; PREGNYL(trademark), Organon) and by purified hCG (CR127) over a concentration range of 0.05 IU to 1,000 IU/ml. Results are shown at day 10. Results with APL(trademark) hCG are indicated by a line with inverted triangles, results with STERIS(trademark) hCG are indicated by a line with circles, results with PREGNYL(trademark) hCG are indicated by a line with squares, and results with hCG-CR127 are indicated by a line with X""s.
FIGS. 5A-C. Effect of preparations of hCG on HIV-LTR activity. (A) The CAT activity of cells containing the HIV-LTR construct and treated with hCG concentrations of 0 IU/ml, 100 IU/ml, 250 IU/ml and 500 IU/ml was calculated relative to the untreated control. (B) Relative CAT activity of the unrelated SV40 promoter in response to hCG at 0 IU/ml, 250 IU/ml and 500 IU/ml was similarly calculated. Data in both A and B represents the mean+/xe2x88x92S.E.M. of 3 to 7 independent experiments and is presented as a bar graph. The different hCG concentrations are indicated in both A and B as depicted below: 
(C) The viability of Hut 78 cells in the presence of hCG at 0 IU/ml, 250 IU/ml and 500 IU/ml under the conditions used in the transient expression assays shown in Figures A and B was determined using a coulter cell counter. Results are presented as a bar graph in terms of cell numberxc3x9710,000 and the hCG concentrations are indicated as shown below: 
FIGS. 6A-H. Effect of hCG preparations and peptides on KS colony growth in vitro and KS tumors in vivo. (A) Comparison of the anti-KS in vitro effects (tumor cell killing) of purified hCG and xcex2-hCG peptides in KS clonogenic assays using KS Y-12 and KS Y-12 xe2x80x9cSKLxe2x80x9d 18 cells depicted in a bar graph in terms of number of colonies. The results are averages of 3 sets of results with less than 10% variation and are representative of multiple experiments. Results with no hCG or hCG peptides are represented by open bars, the results with the xcex2-hCG peptide of amino acids 109-119 (SEQ ID NO:7) are represented by stippled bars, the results with the xcex2-hCG peptide of amino acids 109-145 (SEQ ID NO:25) are represented by the bars with horizontal stripes, the results with the circular xcex2-hCG peptide of amino acids 44-57 (SEQ ID NO:26) where the amino acid at position 44 is a cysteine are represented by the bars with diagonal stripes, and the results with the highly purified hCG preparation, CR 127, are represented by solid bars. (B-H) Thin sections of KS tumors induced in nude mice by inoculation with 106 neoplastic KS Y-1 cells. (B) Thin section of tumors from mice that were not treated with hCG or hCG subunits or peptides (C) Thin section of a tumor from a mouse after treatment with crude hCG APL(trademark) (100 IU) subcutaneously daily for 7 days. (D) Thin section of a tumor from a mouse treated with the xcex2-hCG peptide of amino acids 45-57 (SEQ ID NO:6), 10 xcexcg/ml/daily (6.7 nmoles) for 5 days. (E) Thin section of a tumor from a mouse after 5 days of treatment with the circularized xcex2-hCG peptide 44-57 where cysteine has been substituted at position 44 (SEQ ID NO:26), at 10 xcexcg per day. (F) This panel shows the thin tissue section of KS tumor from AIDS-KS patients treated with 1 ml of diluent alone shows less than 2% cell death as detected by specific apoptosis in situ immunostaining. (G) Thin tissue section of KS tumor from an AIDS-KS patient after hCG preparation therapy of intralesional injections of 2000 IU, 3 times weekly for 2-3 weeks, shows evidence of apoptosis in all cells. (H) Thin tissue section of KS tumor from an AIDS-KS patient after hCG preparation therapy, 500 IU, 3 times weekly for 3 weeks.
FIGS. 7A-C. These bar graphs demonstrate the effects of hCG preparations and peptides on hematopoiesis in vitro. (A) This bar graph depicts results of colony assays in terms of number of colonies for CFU-MIX (colony forming units of megakaryocytes, erythrocytes, granulocytes and monocytes). (B) This bar graph presents data from colony assays for BFU-e (Burst forming units of erythrocytes) in terms of number of colonies. (C) This bar graph presents results from colony assays of CFU-GM (colony forming units of granulo-macrophages) in terms of number of colonies. For all three graphs, results are shown for cells isolated from cord blood (xe2x80x9ccordxe2x80x9d) and bone marrow (xe2x80x9cmarrowxe2x80x9d). The results are averages of 3 sets of results with less than 10% variation and are representative of multiple experiments. The results from no treatment are indicated by open bars; the results with xcex1-hCG are represented by solid bars; the results with APL(trademark) hCG (hCGapl) are represented by bars with a lattice pattern; the results with native xcex2-hCG preparation (NbhCG) are represented by cross-hatched bars; the results with the highly purified hCG preparation (CR127) are represented by open bars; the results with the xcex2-hCG peptide of amino acids 109-119 (SEQ ID NO:7) (b109-119) are shown by the diagonally stippled bars; the results with the xcex2-hCG peptide of amino acids 45-57 (SEQ ID NO:6) (b45-57) are shown by the bars with the diamonds; the results with the circularized xcex2-hCG peptide of amino acids 44-57 with cysteine substituted for the amino acid at position 44 (SEQ ID NO:26) (b45-57c) are represented by the diagonally striped bars; and the results with the mixture of scrambled xcex2-hCG peptides of amino acids 45-57 and 109-119 (bmix45+109) are represented by the vertically striped bars.
FIG. 8. Nucleotide (SEQ 1D NO:1) and amino acid (SEQ 1D NO:2) sequence of xcex2-hCG.
FIGS. 9A and B. Schematic depiction of the structures of (A) the linear peptide of amino acids 45-57 (SEQ ID NO:6) of the xcex2-hCG sequence depicted in FIG. 8 (SEQ ID NO:2) where the amino acid residues at positions 47 and 51 are substituted by a branch made up of diaminobutyric acid peptide bonded to proline, and (B) the circularized peptide of amino acids 44-57 (SEQ ID NO:26) with valine at position 44 substituted with cysteine, which protein is circularized via a disulfide bond between its amino- and carboxy-terminal cysteines. In both A and B, amino acids are represented by their three letter amino acid code, except for the branched residues and the terminal cysteines, for which the structure is depicted.
FIGS. 10A-F. These graphs depict results from the fractionation by Superdex 200 gel filtration of a commercial hCG preparation APL(trademark) (Wyeth Ayerst) and early pregnancy urine. (A) and (D). These graphs depicts the relative amount of protein in mg/ml in each fraction identified by fraction number in the hCG APL(trademark) fractionation (A) and early pregnancy urine fractionation (D). The fractions containing the hCG dimer and xcex2-core protein are identified with arrows and the labels xe2x80x9chCGxe2x80x9d and xe2x80x9cxcex2-corexe2x80x9d respectively. (B) and (E). These graphs present the percent inhibition of growth of cultured KS cells by the individual fractions from the hCG APL(trademark) (B) and early pregnancy urine (E) using KS cell clonogenic assays. The results are plotted as percent inhibition versus fraction number. (C) and (F). These graphs depict the effect of the hCG APL(trademark) (C) and early pregnancy urine (F) fractions on HIV replication in vitro. Specifically, this graph presents data on the percentage inhibition of HIV-1 IIIB viral infection of PBMCs as a function of fraction number.
FIG. 11. Effect of hCG preparations, hCG and early pregnancy urine fractions, and xcex2-hCG peptides on KS cell growth in vitro. Results for clonogenic assays using the cell lines KS Y-1 (bars labeled xe2x80x9cKS Y-1xe2x80x9d) and KS SLK (labeled xe2x80x9cKS SLKxe2x80x9d) are presented on a bar graph as percent inhibition of KS Colony Growth. Bars 1-4 represent cells treated with 200 IU/ml of the commercial hCG preparations hCG APL(trademark), hCG CG10 (Sigma), hCG PROFASI(trademark), and hCG PREGNYL(trademark), respectively; bar 5 represents treatment with 50 xcexcg/ml xcex2-hCG core protein; bar 6, 50 xcexcg/ml native xcex2-hCG; bar 7, 50 xcexcg/ml native xcex1-hCG; bar 8, 200 IU/ml highly purified hCG preparation CR 127; bar 9, 50 xcexcg/ml recombinantly produced hCG (Sigma); bars 10 and 11, 50-100 xcexcl/ml of fractions 65 and 76, respectively, of the early pregnancy urine fractionation; bars 12 and 13, 50-100 xcexcl/ml of fractions 65 and 76, respectively, of the hCG APL(trademark) fractionation; and bar 14, 100 xcexcg/ml of the circularized xcex2-hCG peptide 44-57 (with cysteine substituted at position 44; SEQ ID NO:26).
FIGS. 12A-C. These bar graphs demonstrate the effects of hCG preparations, fractions and peptides on hematopoiesis in vitro. (A) Results of colony assays in terms of percent increase of hematopoiesis for CFU-GEMM (colony forming units of megakaryocytes, erythrocytes, granulocytes and monocytes). (B) Data from colony assays for BFU-e (Burst forming units of erythrocytes) in terms of percent increase of hematopoiesis. (C) Results from colony assays of CFU-GM (colony forming units of granulo-macrophages) in terms of percent increase of hematopoiesis. In all three graphs, bar 1 represents results from treatment with PBS alone; bar 2, the results with 100 xcexcg/ml xcex1-hCG; bar 3, with 200 IU/ml APL(trademark) hCG; bar 4, 200 IU/ml of the highly purified hCG preparation CR 127; bar 5, 100 xcexcg/ml native xcex2-hCG preparation; bar 6, 100 xcexcg/ml of the circularized xcex2-hCG peptide of amino acids 44-57 with cysteine substituted for the amino acid at position 44 (SEQ ID NO:26); bar 7, 100 xcexcl/ml of fraction 65 of the hCG APL(trademark) fractionation; bars 8 and 9, 100 xcexcl/ml of fractions 65 and 26, respectively, of the early pregnancy urine fractionation; and bar 10, 100 xcexcg/ml of the xcex2-hCG core protein.
FIG. 13. Effect of treatment with hCG commercial preparation and early pregnancy urine fractions on KS tumors in mice. Results are plotted as percent inhibition of tumor size as compared to control tumors. The open bar represents mice treated with PBS alone; the solid bar with 100 IU per day of hCG APL(trademark); diagonally striped bars with 200 xcexcl per day of fractions 26, 76, 65, and 82 (as labeled on top of the bars) of the early pregnancy urine fractionation (xe2x80x9cHAF-UF#xe2x80x9d); and open bars with 200 xcexcl per day of fractions 62, 65, 74, 76 and 35 (as labeled on top of the bars) of the hCG APL(trademark) fractionation (xe2x80x9cHAF-CF#xe2x80x9d).
FIG. 14. Effect of hCG preparations, peptides and fractions on survival of HIV-1 transgenic mice. Data is plotted on a bar graph as xe2x80x9c% Survival of Tg26 micexe2x80x9d 10 days after birth, each bar representing 4 mice. None of the untreated mice survived. Bar labeled xe2x80x9cahCGxe2x80x9d represents mice treated with 200 xcexcg per day native xcex1-hCG; bar labeled xe2x80x9cCR127xe2x80x9d, with 300 IU per day of the highly purified hCG preparation CR 127; bar 1, 200 xcexcg per day native xcex2-hCG; bar 2, 300 IU per day hCG APL(trademark); bar labeled xe2x80x9crxcex2hCGxe2x80x9d, with 200 xcexcg per day recombinant xcex2-hCG; bar 3, with 50 xcexcg per day LH (leutinizing hormone); bar 4, with 200 xcexcl per day fraction 65 of the hCG APL(trademark) fractionation; bars 5 and 6, 200 xcexcl per day fractions 65 and 76, respectively, of the early pregnancy urine fractionation; bars labeled xe2x80x9cHAF-UF#26 and xe2x80x9cHAF-CF#26xe2x80x9d, 200 xcexcl per day fraction 26 from the early pregnancy urine and hCG APL(trademark) fractionations, respectively; bar labelled xe2x80x9cb-corexe2x80x9d, 50 xcexcg per day xcex2-hCG core peptide; and bar 7, with 300 xcexcg per day circularized xcex2-hCG peptide 44-57 (with cysteine substituted at position 44; SEQ ID NO:26).
FIGS. 15A-C. Graphs of change in viral load and CD4+ T cell levels with hCG therapy. (A) The change in viral load is plotted as the logarithm of viral load after therapy (xe2x80x9cLogloadxe2x80x9d) as a function of viral load before therapy (xe2x80x9cBaselogxe2x80x9d). (B) The change in CD4+ T cell levels is plotted as CD4+ T cell levels after therapy (in CD4+ T cells/ml) (xe2x80x9cCD4xe2x80x9d) as a function of CD4+ T cell levels before therapy (in CD4+ T cells/ml) (xe2x80x9cCD4Basexe2x80x9d). (C) Plot of linear regression analysis of the change in viral load (xe2x80x9cvlchangexe2x80x9d) as a function of weekly dose of hCG in IU (xe2x80x9cHCGIUxe2x80x9d). For all three panels, data points for patients on hCG therapy as well as non-protease and protease inhibitors are represented by open triangles, those on hCG therapy and non-protease inhibitors by open diamonds, and those on hCG alone by solid circles.
FIGS. 16A and B. (A) Plot of protein concentration (as mAUFS, milli absorbance units, at 280 nm) as a function of the fraction number of the hCG APL(trademark) preparation Superdex 200 fractionation. (B) Plot of protein concentration (as mAUFS, milli absorbance units, at 280 nm) of molecular weight markers of 670 kD, 158 kD, 44 kD, 17 kD and 1.3 kD (as indicated above the plot) as a function of fraction number of a Superdex 200 column run under the same conditions as the fractionation plotted in panel A.
FIGS. 17A-E. Mass spectrometry profiles of fractions 61, 63, 64, 65, and 67 in panels A-E, respectively.
FIGS. 18 A-H. Visualization by confocal microscopy of apoptosis of prostate cancer cells treated with hCG and hCG related preparations for 48 hours and stained with both actin monoclonal antibody labeled with FITC and propidium iodine. Panel A shows a confocal micrograph of cultured prostate tumor cells untreated; Panel B, prostate tumor cells treated with 200 IU hCG APL(trademark); Panel C, prostate tumor cells treated with 200 xcexcg of the circularized xcex2-hCG peptide of amino acids 44-57 with cysteine substituted at position 44 (SEQ ID NO:26); Panel D, prostate tumor cells treated with 100 xcexcl of fraction number 64 of the early pregnancy urine; Panels E-H are controls treated with PBS alone.
FIG. 19. Results of clonogenic assays on prostate tumor cells with hCG preparations and fractions of the hCG APL(trademark) preparations. Results are plotted both as number of colonies (bars) and percent inhibition of colony formation (line with solid diamonds) for each test substance. xe2x80x9cPBSxe2x80x9d are cells treated with PBS alone; xe2x80x9capl100xe2x80x9d and xe2x80x9capl200xe2x80x9d are cells treated with 100 IU and 200 IU of the hCG APL(trademark) preparation, respectively; the numbers are cells treated with those fraction numbers of the fractionation of the hCG APL(trademark) preparation; xe2x80x9calphaxe2x80x9d are those cells treated with a native xcex1-hCG preparation; and xe2x80x9capl-dxe2x80x9d are those cells treated with the hCG APL(trademark) diluent alone (i.e., a control without hCG).
FIGS. 20A-C. Photographs showing the levels of apoptosis in tumor cells in nude mice in response to treatment with hCG preparations or xcex2-hCG peptides. Panel A presents tumor cells from untreated mice. Panel B presents tumor cells from mice treated with 100 xcexcg/day of the circularized xcex2-hCG peptide of amino acids 44-57 with cysteine substituted at position 44 (SEQ ID NO:26). Panel C presents tumor cells from mice treated with 100 IU per day of hCG APL(trademark).
FIG. 21. Bar graph depicting the percent of dead lung cancer cells as measured by Trypan Blue assay in response to hCG preparations, hCG fractions, and xcex2-hCG peptides. Treatments are indicated as follows: xe2x80x9cneg/pbs100xe2x80x9d is control treated with PBS alone; xe2x80x9capl/200xe2x80x9d and xe2x80x9capl/500xe2x80x9d were treated with 200 IU and 500 IU, respectively, of the hCG APL(trademark) preparation; xe2x80x9csata2/200xe2x80x9d and xe2x80x9csata2/300xe2x80x9d were treated with 200 xcexcg/ml and 300 xcexcg/ml, respectively, of the circularized xcex2-hCG peptide of amino acids 44-57, with cysteine substituted for position 44 (SEQ ID NO:26); xe2x80x9cuf-60/200xe2x80x9d, xe2x80x9cuf-64/200xe2x80x9d, xe2x80x9cuf-74/200xe2x80x9d, xe2x80x9cuf-23/200xe2x80x9d and xe2x80x9cuf-80/200xe2x80x9d were treated with 200 xcexcg/ml of the early pregnancy urine SUPERDEX(trademark) 200 fractions 60, 64, 74, 23, and 80, respectively; xe2x80x9capl-64/200xe2x80x9d, xe2x80x9capl-65/200xe2x80x9d, xe2x80x9capl-67/200xe2x80x9d, xe2x80x9capl-72/200xe2x80x9d, xe2x80x9capl-74/200xe2x80x9d and xe2x80x9capl-75/200xe2x80x9d were treated with 200 xcexcg/ml of the hCG APL(trademark) SUPERDEX(trademark) 200 fractions 64, 65, 67, 72, 74, and 75, respectively.
FIGS. 22A-C. Results of clonogenic assays on lung tumor cells with hCG preparations, fractions of hCG preparations, and xcex2-hCG peptides. Results are plotted both as number of colonies (bars) and percent inhibition of colony formation (line with solid diamonds) for each test substance. (A) Treatments are indicated as follows: xe2x80x9c100 ul PBSxe2x80x9d is control treated with PBS alone; xe2x80x9cAPL100xe2x80x9d and xe2x80x9cAPL200xe2x80x9d were treated with 100 IU and 200 IU, respectively, of the hCG APL(trademark) preparation; xe2x80x9cSATA2100xe2x80x9d and xe2x80x9cSATA2200xe2x80x9d were treated with 100 xcexcg/ml and 200 xcexcg/ml, respectively, of the circularized xcex2-hCG peptide of amino acids 44-57, with cysteine substituted for position 44 (SEQ ID NO:26); xe2x80x9cuf-60/200xe2x80x9d, xe2x80x9cuf-64/200xe2x80x9d, and xe2x80x9cuf-74/200xe2x80x9d were treated with 200 xcexcg/ml of the early pregnancy urine SUPERDEX(trademark) 200 fractions 60, 64, and 74, respectively; xe2x80x9capl-60/200xe2x80x9d, xe2x80x9capl-64/200xe2x80x9d and xe2x80x9capl-74/200xe2x80x9d were treated with 200 xcexcg/ml of the hCG APL(trademark) SUPERDEX(trademark) 200 fractions 60, 64, and 74, respectively. (B) Results after 6 days of treatment. Treatments are indicated as follows: xe2x80x9c100 ul PBSxe2x80x9d is control treated with PBS alone; xe2x80x9cAPL100xe2x80x9d and was treated with 100 IU of the hCG APL(trademark) preparation; xe2x80x9cSATA100xe2x80x9d was treated with 100 xcexcg/ml of the xcex2-hCG peptide of amino acids 45-57 (SEQ ID NO: 6); xe2x80x9cSATA2100xe2x80x9d was treated with 100 xcexcg/ml of the circularized xcex2-hCG peptide of amino acids 44-57, with cysteine substituted for position 44 (SEQ ID NO:26); and xe2x80x9cSATAB100xe2x80x9d was treated with 100 xcexcg/ml of the fused xcex2-hCG peptide 45-57::109-119 (SEQ ID NO:30). (C) Results after 5 days of treatment. Treatments are indicated as follows: xe2x80x9c100 looul PBSxe2x80x9d is control treated with PBS alone; xe2x80x9cAPL100xe2x80x9d and xe2x80x9cAPL200xe2x80x9d were treated with 100 IU and 200 IU, respectively, of the hCG APL(trademark) preparation; xe2x80x9cSATA2100xe2x80x9d and xe2x80x9cSATA2200xe2x80x9d were treated with 100 xcexcg/ml and 200 xcexcg/ml, respectively, of the circularized xcex2-hCG peptide of amino acids 44-57, with cysteine substituted for position 44 (SEQ ID NO:26); xe2x80x9cuf-60/200xe2x80x9d, xe2x80x9cuf-64/200xe2x80x9d, and xe2x80x9cuf-74/200xe2x80x9d were treated with 200 xcexcg/ml of the early pregnancy urine SUPERDEX(trademark) 200 fractions 60, 64, and 74, respectively; xe2x80x9capl-60/200xe2x80x9d, xe2x80x9capl-64/200xe2x80x9d and xe2x80x9capl-74/200xe2x80x9d were treated with 200 xcexcg/ml of the hCG APL(trademark) SUPERDEX(trademark) 200 fractions 60, 64, and 74, respectively.
FIGS. 23 A-F. Visualization by confocal microscopy of apoptosis of lung cancer cells treated with hCG and hCG related preparations for 48 hours and stained with both actin monoclonal antibody labeled with FITC and propidium iodine. Panel A shows a confocal micrograph of cultured lung tumor cells untreated; Panel B, lung tumor cells treated with 200 IU hCG APL(trademark); Panel C, lung tumor cells treated with 200 xcexcg of the circularized xcex2-hCG peptide of amino acids 44-57 with cysteine substituted at position 44 (SEQ ID NO:26); Panel D, lung tumor cells treated with fraction number 64 of the early pregnancy urine; Panels E-F are controls treated with PBS alone.
FIGS. 24 A-I. Visualization by confocal microscopy of apoptosis of kidney (renal) cancer cells treated with hCG APL(trademark) preparation and stained with both actin monoclonal antibody labeled with FITC and propidium iodine. Panels A-C show a confocal micrograph of cultured kidney cancer cells treated with PBS alone; Panels D-F, cultured kidney cancer cells treated for 48 hours with 100 IU hCG APL(trademark); Panels G-I, cultured kidney cancer cells treated with 300 IU hCG APL(trademark) for 48 hours.
The present invention relates to proteins (including peptides) containing a sequence of one or more portions of xcex2-hCG (xcex2-hCG peptides) that are effective at inhibiting HIV replication and/or infection in vitro or in vivo, decreasing viral load, and/or treating or preventing disorders associated with HIV infection. In specific embodiments, the invention provides an isolated protein, comprising, or alternatively, consisting of an amino acid sequence amino acid numbers 41-54, 45-54, 47-53 or 45-57 (SEQ ID NOS:3-6, respectively) of the xcex2-hCG sequence depicted in FIG. 8 (a portion of SEQ ID NO:2), particularly to an isolated protein or peptide comprising an amino acid sequence consisting of amino acids 45-57 (SEQ ID NO:5) of FIG. 8 (a portion of SEQ ID NO:2). The invention also provides isolated proteins comprising or, alternatively, consisting of, the amino acid sequence of two or more portions (preferably non-naturally contiguous portions) of xcex2-hCG, e.g., wherein such portions are linked at the C-termini and N-termini via peptide bond(s). Specifically, the invention provides isolated proteins having an amino acid sequence of xcex2-hCG amino acids 45-57 (SEQ ID NO:6) linked at the C-terminus via a peptide bond to the N-terminus of amino acids xcex2-hCG 109-119 (SEQ ID NO:7) or linked at the N-terminus via a peptide bond to the C-terminus of xcex2-hCG amino acids 110-119 (SEQ ID NO:27); or an isolated protein of xcex2-hCG amino acids 47-57 (SEQ ID NO:28) linked at the C-terminus via a peptide bond to the N-terminus of amino acids xcex2-hCG 108-119 (SEQ ID NO:29) of the xcex2-hCG sequence depicted in FIG. 8 (portions of SEQ ID NO:2), i.e., the fused peptides denoted 45-57::109-119, 110-119::45-57, or 47-57::108-119 (SEQ ID NOS:30-32, respectively).
The present invention also relates to certain fractions (i.e. components of a source of native hCG or native xcex2-hCG isolated away from other components in the source of native hCG or native xcex2-hCG by a separation technique known in the art), particularly fractions from gel filtration sizing chromatography, of a source of native hCG or xcex2-hCG, such as commercial preparations of hCG and human (preferably early, i.e. first trimester) pregnancy urine, that have anti-HIV and/or anti-KS activity. Native hCG or native xcex2-hCG refers to naturally occurring hCG or xcex2-hCG, i.e. not recombinantly produced.
The present invention further relates to therapeutic methods and compositions for treatment and prevention of disorders associated with HIV infection based on hCG and xcex2-hCG preparations, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG, and therapeutically and prophylactically effective xcex2-hCG peptides. The invention provides for treatment of HIV infection by administration of a therapeutic compound of the invention. The therapeutic compounds of the invention include: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG, therapeutically and prophylactically effective xcex2-hCG peptides, related derivatives and analogs of hCG, xcex2-hCG or xcex2-hCG peptides, and nucleic acids encoding xcex2-hCG and xcex2-hCG peptides, and analogs and derivatives thereof. xcex2-hCG peptides which are effective for treatment and prevention of HIV infection can be identified by in vitro and in vivo assays such as those described herein.
In a preferred embodiment, a therapeutic composition of the invention comprises a xcex2-HCG peptide, the amino acid sequence of which consists of amino acid numbers 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145, 58-145, 109-145, 7-40, 46-65, or 48-56 (SEQ ID NOS:8-25 or 33-35, respectively) of FIG. 8 (a portion of SEQ ID NO:2), particularly a xcex2-hCG peptide which consists of amino acid numbers 41-54, 45-54 or 109-119 (SEQ ID NOS:3, 4, or 7, respectively), most preferably of a xcex2-hCG peptide which consists of amino acid numbers 47-53 (SEQ ID NO:5) or 45-57 (SEQ ID NO:6).
In another preferred embodiment, a therapeutic composition of the invention comprises a fusion protein comprising two or more xcex2-hCG sequences (either as non-contiguous or contiguous sequences), e.g. having an amino acid sequence of one xcex2-hCG peptide linked via a peptide bond to another xcex2-hCG peptide, in particular a protein, the amino acid sequence of which consists of amino acids 45-57 (SEQ ID NO:6) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 109-119 (SEQ ID NO:7) or linked at the N-terminus via a peptide bond to the C-terminus of amino acids 110-119 (SEQ ID NO:27); or an isolated protein of amino acids 47-57 (SEQ ID NO:28) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 108-119 (SEQ ID NO:29) of the xcex2-hCG sequence depicted in FIG. 8 (portions of SEQ ID NO:2), i.e., the peptides denoted 45-57::109-119, 110-119::45-57, or 47-57::108-119 (SEQ ID NOS:30-32, respectively).
In another preferred embodiments, the therapeutic comprises a xcex2-hCG peptide, the amino acid sequence of which consists of circularized (via a disulfide bond between its amino- and carboxy-terminal cysteines) 44-57 (SEQ ID NO:26) with the valine at position 44 substituted with cysteine ((Val44Cys) 45-57 circularized) (depicted in FIG. 9B), the circularized (via a disulfide bond between its amino- and carboxy-terminal cysteines) fused peptide of amino acids 110-119 (SEQ ID NO:27) linked at the C-terminus by a peptide bond to the N-terminus of amino acids 45-57 (SEQ ID NO:6), or the peptide 45-57 (SEQ ID NO:6) where the amino acid residues at positions 47 and 51 are substituted by a branch, where the branches are made up of diaminobutyric acid peptide bonded to a proline residue (depicted in FIG. 9A). The amino acid sequence of xcex2-hCG is depicted in FIG. 8 (SEQ ID NO:2).
In yet another embodiment, the therapeutic comprises fractions, preferably fractions of a source of hCG or xcex2-hCG, such as commercial hCG preparations and human early pregnancy urine, of material eluting from a gel filtration column with apparent molecular weights of approximately 40 kD, 15 kD or 2-3 kD as determined based on in which fractions native hCG dimer (77 kD) and xcex2-hCG core protein (10 kD) elute.
The present invention further relates to therapeutic methods and compositions for treatment and prevention of wasting syndromes based on hCG and xcex2-hCG preparations, therapeutically and prophylactically effective fractions of a source of hCG or xcex2-hCG, and therapeutically and prophylactically effective xcex2-hCG peptides. The invention provides for treatment of wasting syndrome by administration of a therapeutic compound (termed herein xe2x80x9cTherapeuticxe2x80x9d) of the invention. Such Therapeutics of the invention include but are not limited to: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG, therapeutically and prophylactically effective xcex2-hCG proteins (i.e., those peptides which prevent or treat wasting syndrome), related derivatives and analogs of hCG, xcex2-hCG or xcex2-hCG peptides, and nucleic acids encoding xcex2-hCG and xcex2-hCG peptides, and analogs and derivatives thereof. xcex2-hCG peptides which are effective for treatment and prevention of wasting syndromes can be identified by in vitro and in vivo assays such as those described in herein.
The present invention also relates to therapeutic methods and compositions for treatment and prevention of diseases and disorders in which increased amounts of hematopoietic cells are desirable (e.g., disorders associated with reduced numbers of one or more hematopoietic cell types) by administration of hCG, xcex2-hCG and therapeutically or prophylactically effective proteins (e.g., peptides) having a sequence of one or more portions of xcex2-hCG (xcex2-hCG peptides), and derivatives and analogs thereof, and therapeutically or prophylactically effective fractions of a source of native hCG or native xcex2-hCG (i.e. from a naturally occurring source and not hCG or xcex2-hCG which has been recombinantly produced). The invention provides for treatment and prevention of hematopoietic cell deficiencies by administration either of a therapeutic compound of the invention or of hematopoietic cells, the numbers of which have been increased in vitro by contact with a therapeutic compound of the invention. The invention also provides methods for expansion of hematopoietic cells in vitro by contact with a Therapeutic of the invention. The therapeutic compounds of the invention include, but are not limited to: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG, and therapeutically and prophylactically effective xcex2-hCG peptides, i.e., those fractions and peptides which prevent or treat HIV infection (e.g., as demonstrated in in vitro and in vivo assays described infra), and derivatives and analogs thereof, as well as nucleic acids encoding hCG, xcex2-hCG and therapeutically and prophylactically effective xcex2-hCG peptides and derivatives and analogs thereof (e.g., for use in gene therapy).
In a preferred embodiment, a therapeutic composition of the invention comprises a xcex2-hCG peptide, the amino acid sequence of which consists of amino acid numbers 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145, 58-145, 109-145, 7-40, 46-65, or 48-56 (SEQ ID NOS:8-25 or 33-35, respectively) of FIG. 8 (a portion of SEQ ID NO:2), particularly a xcex2-hCG peptide which consists of amino acid numbers 41-54, 45-54 or 109-119 (SEQ ID NOS:3, 4, or 7, respectively), most preferably of a xcex2-hCG peptide which consists of amino acids 47-53 (SEQ ID NO:5) or 45-57 (SEQ ID NO:6).
In other preferred embodiments, the therapeutic comprises a xcex2-hCG peptide, the amino acid sequence of which consists of circularized (via a disulfide bond between its amino- and carboxy-terminal cysteines) 44-57 (SEQ ID NO:26) with the valine at position 44 substituted with cysteine ((Val44Cys) 45-57 circularized) (depicted in FIG. 9B), 45-57 (SEQ ID NO:6) where the amino acid residues at positions 47 and 51 are substituted by a branch, where the branches are made up of diaminobutyric acid peptide bonded to a proline residue (depicted in FIG. 9A).
In another embodiment, a protein is used which contains the amino acid sequence of two or more peptides of different portions of the xcex2-hCG sequence (either as non-contiguous or contiguous sequenced), e.g., in which the N-terminus of one portion is linked to the C-terminus of another portion by peptide bond(s). In a specific embodiment, a protein is used, the amino acid sequence of which consists of amino acids 45-57 (SEQ ID NO:6) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 109-119 (SEQ ID NO:7) or linked at the N-terminus via a peptide bond to the C-terminus of amino acids 110-119 (SEQ ID NO:27); or a protein is used that has an amino acid sequence of amino acids 47-57 (SEQ ID NO:28) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 108-119 (SEQ ID NO:29) of the xcex2-hCG sequence; i.e., the fused peptides represented as 45-57::109-119, 110-119::45-57, or 47-57::108-119 (SEQ ID NOS:30-32, respectively). In yet another embodiment a protein is used which is a cyclic, fused peptide, particularly a cyclic, fused peptide having a sequence consisting of xcex2-hCG amino acids 110-119 (SEQ ID NO:27) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 45-57 (SEQ ID NO:6) and being circularized by a disulfide bond between the terminal cysteines at positions 110 and 57.
In another embodiment, a protein is used that (a) comprises a xcex2-hCG amino acid sequence consisting of amino acid numbers 41-54, 45-54, 47-53, 45-57, 109-119, 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145, 58-145, 109-119, 109-145, 7-40, 46-65 or 48-56 (SEQ ID NOS:3-25 or 33-35, respectively) as depicted in FIG. 8 (a portion of SEQ ID NO:2) and (b) lacks xcex2-hCG amino acids contiguous to said sequence. The amino acid sequence of xcex2-hCG is depicted in FIG. 8 (SEQ ID NO:2).
The present invention further relates to therapeutic methods and compositions for treatment and prevention of cancers based on hCG and xcex2-hCG preparations, therapeutically and prophylactically effective fractions of a source of hCG or xcex2-hCG, and therapeutically and prophylactically effective xcex2-hCG peptides. The invention provides for treatment of cancer by administration of a therapeutic compound (termed herein xe2x80x9cTherapeuticxe2x80x9d) of the invention. Such Therapeutics of the invention include but are not limited to: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG, therapeutically and prophylactically effective xcex2-hCG proteins (i.e., those peptides which prevent or treat cancer), related derivatives and analogs of hCG, xcex2-hCG or xcex2-hCG peptides, and nucleic acids encoding xcex2-hCG and xcex2-hCG peptides, and analogs and derivatives thereof. xcex2-hCG peptides and fractions of a source of native hCG or xcex2-hCG which are effective for treatment and prevention of cancer can be identified by in vitro and in vivo assays such as those described in herein.
In a preferred embodiment, a therapeutic composition of the invention comprises a xcex2-hCG peptide, the amino acid sequence of which consists of amino acid numbers 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145, 58-145, 109-145, 7-40, 46-65, or 48-56 (SEQ ID NOS:8-25 or 33-35, respectively) of FIG. 8 (a portion of SEQ ID NO:2), particularly a xcex2-hCG peptide which consists of amino acid numbers 41-54, 45-54 or 109-119 (SEQ ID NOS:3, 4, or 7, respectively), most preferably of a xcex2-hCG peptide which consists of amino acid numbers 47-53 (SEQ ID NO:5) or 45-57 (SEQ ID NO:6). In another preferred embodiment, a therapeutic composition of the invention comprises a fusion protein comprising more than one xcex2-hCG sequence (preferably non-contiguous sequences), e.g. having an amino acid sequence of one xcex2-hCG peptide linked via a peptide bond to another xcex2-hCG peptide, in particular a protein, the amino acid sequence of which consists of amino acids 45-57 (SEQ ID NO:6) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 109-119 (SEQ ID NO:7) or linked at the N-terminus via a peptide bond to the C-terminus of amino acids 110-119 (SEQ ID NO:27); or an isolated protein of amino acids 47-57 (SEQ ID NO:28) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 108-119 (SEQ ID NO:29) of the xcex2-hCG sequence depicted in FIG. 8 (portions of SEQ ID NO:2), i.e., the peptides denoted 45-57::109-119, 110-119::45-57, or 47-57::108-119 (SEQ ID NOS:30-32, respectively).
In other preferred embodiments, the therapeutic comprises a xcex2-hCG peptide, the amino acid sequence of which consists of circularized (via a disulfide bond between its amino- and carboxy-terminal cysteines) 44-57 (SEQ ID NO:26) with the valine at position 44 substituted with cysteine ((Val44Cys) 45-57 circularized) (depicted in FIG. 9B), the circularized (via a disulfide bond between its amino- and carboxy-terminal cysteines) fused peptide of amino acids 110-119 (SEQ ID NO:27) linked at the C-terminus by a peptide bond to the N-terminus of amino acids 45-57 (SEQ ID NO:6), or the peptide 45-57 (SEQ ID NO:6) where the amino acid residues at positions 47 and 51 are substituted by a branch, where the branches are made up of diaminobutyric acid peptide bonded to a proline residue (depicted in FIG. 9A). The amino acid sequence of xcex2-hCG is depicted in FIG. 8 (SEQ ID NO:2).
In yet another embodiment, the therapeutic comprises fractions, preferably gel filtration fractions of a source of native hCG or native xcex2-hCG (i.e. from or derived from a naturally occurring source of hCG or xcex2-hCG and not recombinantly produced hCG or xcex2-hCG), such as commercial hCG preparations and human pregnancy (preferably early, i.e. first trimester) urine, of material eluting from a SUPERDEX(trademark) 200 (Pharmacia) gel filtration column with apparent molecular weights of approximately 40 kD, 15 kD or 2-3 kD as determined based on in which fractions native hCG dimer (77 kD) and xcex2-hCG core (10 kD) elute.
For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections which follow.
xcex2-hCG Peptides and Derivatives Thereof
The invention provides isolated proteins (e.g., peptides), the amino acid sequences of which consist of one or more portions of the xcex2-hCG sequence (xcex2-hCG peptides), and derivatives thereof, which are effective for treatment or prevention of HIV infection and resulting disorders. In various specific embodiments, the portions of the xcex2-hCG sequence are at least 3, 5, 10, 20, or 30 amino acids. Effectiveness of the peptides of the invention for treatment or prevention of HIV infection can be determined by any of the methods disclosed herein or by any method known in the art. In a specific embodiment, the peptides inhibit HIV infection or replication. In a preferred embodiment, the invention relates to proteins, the amino acid sequences of which consist of amino acid numbers 41-54, 45-54, 47-53 and 45-57 (SEQ ID NOS:3-6) of the xcex2-hCG sequence depicted in FIG. 8 (a portion of SEQ ID NO:2). In other embodiments, proteins, the amino acid sequences of which consist of amino acid numbers 109-119, 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145; 58-145, 109-145, 7-40, 46-65, and 48-56 (SEQ ID NOS:7-25 and 33-35, respectively) of FIG. 8 (a portion of SEQ ID NO:2) are also provided.
In another embodiment, the invention provides proteins, the amino acid sequences of which consist of two or more at least 5, 7 or 10 amino acid, non-naturally contiguous portions of the xcex2-hCG sequence (FIG. 8 (SEQ ID NO:2)) linked by peptide bonds between the N-terminus of one portion and the C-terminus of another portion. Specifically, proteins, the amino acid sequences of which consist of amino acids 45-57 (SEQ ID NO:6) linked at the C-terminus via a peptide bond to the N-terminus of a peptide of amino acids 109-119 (SEQ ID NO:7) or linked at the N-terminus via a peptide bond to the C-terminus of amino acids 110-119 (SEQ ID NO:27); or an isolated protein of amino acids 47-57 (SEQ ID NO:28) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 108-119 (SEQ ID NO:29) of the xcex2-hCG sequence depicted in FIG. 8 (portions of SEQ ID NO:2), i.e., the fused peptides denoted as 45-57::109-119, 110-119::45-57, or 47-57::108-119 (SEQ ID NOS:30-32, respectively). Derivatives of the foregoing fusion proteins are also provided (e.g., branched, cyclized, N- or C-terminal chemically modified, etc.). In another embodiment, fusion proteins comprising two or more such portions of the xcex2-hCG sequence are provided; such portions may or may not be contiguous to one another (i.e., an intervening sequence may be present). Molecules comprising such portions linked by hydrocarbon linkages are also provided. In another embodiment, the peptides of the invention (i) have an amino acid sequence consisting of no more than 8 peptides of the xcex2-hCG sequence (FIG. 8 (SEQ ID NO:2)) and (ii) comprise amino acid numbers 47-53 (SEQ ID NO:5) of xcex2-hCG (FIG. 8 (SEQ ID NO:2)).
In another embodiment, the invention provides an isolated protein which protein (a) comprises one or more portions of the amino acid sequence of xcex2-hCG, a peptide consisting of said portion(s) being effective to inhibit HIV infection or replication; and (b) lacks xcex2-hCG amino acids contiguous to said portion(s). In a specific embodiment, the invention provides an isolated protein (a) comprising a xcex2-hCG amino acid sequence consisting of amino acid numbers 41-54, 45-54, 47-53, 45-57, 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-56, 47-58, 58-145, 7-40, 46-65 or 48-56 (SEQ ID NOS:3-6, 8-19, 21, 22, 24, or 33-35, respectively) as depicted in FIG. 8 (a portion of SEQ ID NO:2); and (b) lacking xcex2-hCG amino acids contiguous to said sequence. Peptides containing the above sequences in which only conservative substitutions have been made are also provided by the present invention, as but one example of peptide derivatives within the scope of the invention. Analogs of the above-mentioned proteins and peptides which have one or more amino acid substitutions forming a branched peptide (e.g., by substitution with an amino acid or amino acid analog having a free amino- or carboxy-side chain that forms a peptide bond with a sequence of one or more amino acids, including but not limited to prolines) or allowing circularization of the peptide (e.g., by substitution with a cysteine, or insertion of a cysteine at the amino- or carboxy-terminus or internally), to provide a sulfhydryl group for disulfide bond formation, are also provided.
In specific embodiments, peptides of less than 50, or less than 25, amino acids are provided.
The invention also relates to derivatives, modifications and analogs of xcex2-hCG peptides. In one embodiment, xcex2-hCG peptide derivatives can be made by altering the xcex2-hCG peptide sequence by substitutions, additions or deletions that provide for therapeutically effective molecules. Thus, the xcex2-hCG peptide derivatives include peptides containing, as a primary amino acid sequence, all or part of the particular xcex2-hCG amino acid sequence including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a peptide which is functionally active. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
Conservative substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such xcex2-hCG peptide derivatives can be made either by chemical peptide synthesis or by recombinant production from nucleic acid encoding the xcex2-hCG peptide which nucleic acid has been mutated. Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem 253:6551), use of TAB(copyright) linkers (Pharmacia), etc.
In addition, xcex2-hCG peptides and analogs and derivatives of xcex2-hCG peptides can be chemically synthesized (see, e.g., Merrifield, 1963, J. Amer. Chem. Soc. 85:2149-2156). For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., see Creighton, 1983, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., N.Y., pp. 50-60). xcex2-hCG peptides can also be synthesized by use of a peptide synthesizer. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., N.Y., pp. 34-49). Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the xcex2-hCG peptide. Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, xcex1-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, xcex3-Abu, xcex5-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, xcex2-alanine, fluoro-amino acids, designer amino acids such as xcex2-methyl amino acids, Cxcex1-methyl amino acids, Nxcex1-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).
By way of example but not by way of limitation, peptides of the invention can be chemically synthesized and purified as follows: Peptides can be synthesized by employing the N-xcex1-9-fluorenylmethyloxycarbonyl or Fmoc solid phase peptide synthesis chemistry using a Rainin Symphony Multiplex Peptide Synthesizer. The standard cycle used for coupling of an amino acid to the peptide-resin growing chain generally includes: (1) washing the peptide-resin three times for 30 seconds with N,N-dimethylformamide (DMF); (2) removing the Fmoc protective group on the amino terminus by deprotection with 20% piperdine in DMF by two washes for 15 minutes each, during which process mixing is effected by bubbling nitrogen through the reaction vessel for one second every 10 seconds to prevent peptide-resin settling; (3) washing the peptide-resin three times for 30 seconds with DMF; (4) coupling the amino acid to the peptide resin by addition of equal volumes of a 250 mM solution of the Fmoc derivative of the appropriate amino acid and an activator mix consisting or 400 mM N-methylmorpholine and 250 mM (2-(1H-benzotriazol-1-4))-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) in DMF; (5) allowing the solution to mix for 45 minutes; and (6) washing the peptide-resin three times for 30 seconds of DMF. This cycle can be repeated as necessary with the appropriate amino acids in sequence to produce the desired peptide. Exceptions to this cycle program are amino acid couplings predicted to be difficult by nature of their hydrophobicity or predicted inclusion within a helical formation during synthesis. For these situations, the above cycle can be modified by repeating step 4 a second time immediately upon completion of the first 45 minute coupling step to xe2x80x9cdouble couplexe2x80x9d the amino acid of interest. Additionally, in the first coupling step in peptide synthesis, the resin can be allowed to swell for more efficient coupling by increasing the time of mixing in the initial DMF washes to three 15 minute washes rather than three 30 second washes.
After peptide synthesis, the peptide can be cleaved from the resin as follows: (1) washing the peptide-resin three times for 30 seconds with DMF; (2) removing the Fmoc protective group on the amino terminus by washing two times for 15 minutes in 20% piperdine in DMF; (3) washing the peptide-resin three times for 30 seconds with DMF; and (4) mixing a cleavage cocktail consisting of 95% trifluoroacetic acid (TFA), 2.4% water, 2.4% phenol, and 0.2% triisopropysilane with the peptide-resin for two hours, then filtering the peptide in the cleavage cocktail away from the resin, and precipitating the peptide out of solution by addition of two volumes of ethyl ether.
To isolate the peptide, the ether-peptide solution can be allowed to sit at xe2x88x9220xc2x0 C. for 20 minutes, then centrifuged at 6,000xc3x97G for 5 minutes to pellet the peptide, and the peptide can be washed three times with ethyl ether to remove residual cleavage cocktail ingredients. The final peptide product can be purified by reversed phase high pressure liquid chromatography (RP-HPLC) with the primary solvent consisting of 0.1% TFA and the eluting buffer consisting of 80% acetonitrile and 0.1% TFA. The purified peptide can then be lyophilized to a powder.
In a preferred embodiment, the invention provides a peptide with branched amino acids (branched peptide), preferably a branched peptide of amino acids 45-57 (SEQ ID NO:6) with branches occurring at positions 47 and 51, respectively, instead of the Gly and Ala residues normally present. Most preferably, diaminobutyric acid is substituted for the gly and ala residues at positions 47 and 51, respectively, and proline bonded to both diaminobutyric acid residues (45-57 branched) as shown in FIG. 9A.
In other specific embodiments, branched versions of the xcex2-hCG peptides listed hereinabove are provided, e.g., by substituting one or more amino acids within the xcex2-hCG sequence with an amino acid or amino acid analog with a free side chain capable of forming a peptide bond with one or more amino acids (and thus capable of forming a xe2x80x9cbranchxe2x80x9d).
Branched peptides may be prepared by any method known in the art for covalently linking any naturally occurring or synthetic amino acid to any naturally occurring or synthetic amino acid in a peptide chain which has a side chain group able to react with the amino or carboxyl group on the amino acids so as to become covalently attached to the peptide chain. In particular, amino acids with a free amino side chain group, such as, but not limited to, diaminobutyric acid, lysine, arginine, ornithine, diaminopropionic acid and citrulline, can be incorporated into a peptide so that an amino acid can form a branch therewith, for example, by forming a peptide bond to the free amino side group, from that residue. Alternatively, amino acids with a free carboxyl side chain group, such as, but not limited to, glutamic acid, aspartic acid and homocitrulline, can be incorporated into the peptide so that an amino acid can form a branch therewith, for example, by forming a peptide bond to the free carboxyl side group, from that residue. The amino acid forming the branch can be linked to a side chain group of an amino acid in the peptide chain by any type of covalent bond, including, but not limited to, peptide bonds, ester bonds and disulfide bonds. In a specific embodiment, amino acids, such as those described above, that are capable of forming a branch point, are substituted for xcex2-hCG residues within a peptide having a xcex2-hCG sequence.
Branched peptides can be prepared by any method known in the art. For example, but not by way of limitation, branched peptides can be prepared as follows: (1) the amino acid to be branched from the main peptide chain can be purchased as an N-xcex1-tert-butyloxycarbonyl (Boc) protected amino acid pentafluorophenyl (Opfp) ester and the residue within the main chain to which this branched amino acid will be attached can be an N-Fmoc-xcex1-xcex3-diaminobutyric acid; (2) the coupling of the Boc protected amino acid to diaminobutyric acid can be achieved by adding 5 grams of each precursor to a flask containing 150 ml DMF, along with 2.25 ml pyridine and 50 mg dimethylaminopyridine and allowing the solution to mix for 24 hours; (3) the peptide can then be extracted from the 150 ml coupling reaction by mixing the reaction with 400 ml dichlormethane (DCM) and 200 ml 0.12 N HCl in a 1 liter separatory funnel, and allowing the phases to separate, saving the bottom aqueous layer and re-extracting the top layer two more times with 200 ml 0.12 N HCl; (4) the solution containing the peptide can be dehydrated by adding 2-5 grams magnesium sulfate, filtering out the magnesium sulfate, and evaporating the remaining solution to a volume of about 2-5 ml; (5) the dipeptide can then be precipitated by addition of ethyl acetate and then 2 volumes of hexanes and then collected by filtration and washed two times with cold hexanes; and (6) the resulting filtrate can be lyophilized to achieve a light powder form of the desired dipeptide. Branched peptides prepared by this method will have a substitution of diaminobutyric acid at the amino acid position which is branched. Branched peptides containing an amino acid or amino acid analog substitution other than diaminobutyric acid can be prepared analogously to the procedure described above, using the N-F-moc coupled form of the amino acid or amino acid analog.
In a preferred embodiment, the peptide is a cyclic peptide, preferably a cyclic peptide of xcex2-hCG amino acids 44-57 with cysteine substituted for valine at position 44 (SEQ ID NO:26) and circularized via a disulfide bond between the cysteine residues at positions 44 and 57 (C[V44C] 45-57) (FIG. 9B), or a cyclic fused peptide of xcex2-hCG amino acids 110-119 (SEQ ID NO:27) linked at the C-terminus by a peptide bond to the N-terminus of amino acids 45-57 (SEQ ID NO:6) and circularized via a disulfide bond between the cysteine residues at positions 110 and 57. In another preferred embodiment, the peptide is a cyclic branched peptide of xcex2-hCG amino acids 44-57 (SEQ ID NO:26) with cysteine substituted for valine at position 44 and circularized via a disulfide bond between the cysteine residues at positions 44 and 57 and positions 47 and 51 substituted with a diaminobutyric acid residue on which a proline is peptide bonded to its free amino sidechain.
Cyclization can be, for example but not by way of limitation, via a disulfide bond between two cysteine residues or via an amide linkage. For example, but not by way of limitation, disulfide bridge formation can be achieved by (1) dissolving the purified peptide at a concentration of between 0.1.-0.5 mg/ml in 0.01 M ammonium acetate, pH 7.5; (2) adding 0.01 M potassium ferricyanide to the dissolved peptide dropwise until the solution appears pale yellow in color and allowing this solution to mix for 24 hours; (3) concentrating the cyclized peptide to 5-10 ml of solution, repurifying the peptide by reverse phase-high pressure liquid chromatography (RP-HPLC) and finally lyophilizing the peptide. In a specific embodiment, in which the peptide does not contain two appropriately situated cysteine residues, cysteine residues can be introduced at the amino-terminus and/or carboxy-terminus and/or internally such that the peptide to be cyclized contains two cysteine residues spaced such that the residues can form a disulfide bridge.
Alternatively, a cyclic peptide can be obtained by generating an amide linkage. An amide linkage can be achieved by, for example, but not limited to, the following procedure: An allyl protected amino acid, such as aspartate, glutamate, asparagine or glutamine, can be incorporated into the peptide as the first amino acid, and then the remaining amino acids coupled on. The allyl protective group can be removed by a two hour mixing of the peptide-resin with a solution of tetrakistriphenylphophine palladium (0) in a solution of chloroform containing 5% acetic acid and 2.5% N-methylmorpholine. The peptide resin can be washed three times with 0.5% N,N-diisopropylethylamine (DIEA) and 0.5% sodium diethyldithiocabamate in DMF. The amino terminal Fmoc group on the peptide chain can be removed by two incubations for 15 minutes each in 20% piperdine in DMF, and washed three times with DMF for 30 seconds each. The activator mix, N-methylmorpholine and HBTU in DMF, can be brought onto the column and allowed to couple the free amino terminal end to the carboxyl group generated by removal of the allyl group to cyclize the peptide. The peptide can cleaved from the resin as described in the general description of chemical peptide synthesis above and the peptide purified by reverse phase-high pressure liquid chromatography (RP-HPLC). In a specific embodiment, in which the peptide to be cyclized does not contain an allyl protected amino acid, an allyl protected amino acid can be introduced into the sequence of the peptide, at the amino-terminus, carboxy-terminus or internally, such that the peptide can be cyclized.
xcex2-hCG peptides can also be obtained by recombinant expression techniques. (See, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 2d Ed., Cold Spring Harbor, N.Y., Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K., Vol. I, II). The nucleic acid sequence encoding hCG has been cloned and the sequence determined (FIG. 8 (SEQ ID NOS:1 and 2) and Xia, H., 1993, J. Molecular Endocrinology Jun. 10, 1993:337-343; Sherman, G. B., 1992, J. Molecular Endocrinology, Jun. 6, 1992:951-959; Gieseman, L. K. (ed.), 1991, Basic and Chemical Endocrinology, pp. 543-567; Ward et al., 1991, in Reproduction in Domestic Animals, 4th ed., P. T. Coppos, ed., pp. 25-80, Academic Press, New York) and can be isolated using well-known techniques in the art, such as screening a library, chemical synthesis, or polymerase chain reaction (PCR).
To recombinantly produce a xcex2-hCG peptide, a nucleic acid sequence encoding xcex2-hCG or a xcex2-hCG peptide is operatively linked to a promoter such that xcex2-hCG or a xcex2-hCG peptide is produced from said sequence. For example, a vector can be introduced into a cell, within which cell the vector or a portion thereof is expressed, producing xcex2-hCG or one or more portions thereof. In a preferred embodiment, the nucleic acid is DNA if the source of RNA polymerase is DNA-directed RNA polymerase, but the nucleic acid may also be RNA if the source of polymerase is RNA-directed RNA polymerase or if reverse transcriptase is present in the cell or provided to produce DNA from the RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in bacterial or mammalian cells. Expression of the sequence encoding xcex2-hCG or the xcex2-hCG peptide can be by any promoter known in the art to act in bacterial or mammalian cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3xe2x80x2 long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the HSV-1 (herpes simplex virus-1) thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42), etc., as well as the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene control region which is active in erythroid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46, 89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropin releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378). The promoter element which is operatively linked to the nucleic acid encoding xcex2-hCG or a xcex2-hCG peptide can also be a bacteriophage promoter with the source of the bacteriophage RNA polymerase expressed from a gene for the RNA polymerase on a separate plasmid, e.g., under the control of an inducible promoter, for example, the nucleic acid encoding xcex2-hCG or xcex2-hCG peptide operatively linked to the T7 RNA polymerase promoter with a separate plasmid encoding the T7 RNA polymerase.
In a less preferred embodiment, peptides can be obtained by proteolysis of hCG or xcex2-hCG followed by purification using standard techniques such as chromatography (e.g., HPLC), electrophoresis, etc.
Also included within the scope of the invention are xcex2-hCG peptide derivatives which are differentially modified during or after synthesis, e.g., by benzylation, glycosylation, acetylation, phosphorylation, amidation, pegylation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. In specific embodiments, the peptides are acetylated at the N-terminus and/or amidated at the C-terminus.
Any of numerous chemical modifications may be carried out by known techniques, including but not limited to acetylation, formulation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
In another embodiment, the xcex2-hCG or xcex2-hCG peptide derivative is a chimeric, or fusion, protein comprising xcex2-hCG or a functional xcex2-hCG peptide (or two or more portions of xcex2-hCG joined by peptide bond(s)) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein. In a specific embodiment, the derivative is a fusion protein comprising the xcex2-hCG sequence, or portions thereof, joined at its amino or carboxy-terminus to an amino acid sequence, or portions thereof, of a chemokine which is therapeutically useful in the treatment of AIDS, for example, the chemokines MIP-1xcex1, MIP-1xcex2 or Rantes (for amino acid sequences of these chemokines see Shall, 1991, Cytokine 3:165-183). In one embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a xcex2-hCG-coding sequence joined in-frame to a coding sequence for a different protein). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
Therapeutic Uses
HIV
The invention provides for treatment or prevention of diseases and disorders associated with HIV infection by administration of a therapeutic compound (termed herein xe2x80x9cTherapeuticxe2x80x9d). Such xe2x80x9cTherapeuticsxe2x80x9d include, but are not limited to: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG, and therapeutically and prophylactically effective xcex2-hCG peptides, i.e., those fractions and peptides which prevent or treat HIV infection (e.g., as demonstrated in in vitro and in vivo assays described infra), and derivatives and analogs thereof, as well as nucleic acids encoding hCG, xcex2-hCG and therapeutically and prophylactically effective xcex2-hCG peptides and derivatives and analogs thereof (e.g., for use in gene therapy). Examples of Therapeutics are those proteins described herein and nucleic acids encoding such proteins and frations of native hCG and native xcex2-hCG described below.
A preferred embodiment of the invention relates to methods of using a Therapeutic for treatment or prevention of HIV infection, preferably HIV-1 infection, in a human subject. In a specific embodiment, the Therapeutic is used for the treatment or prevention of HIV infection in a human subject that does not suffer from a cancer which secretes hCG or hCG fragments. In another specific embodiment, the Therapeutic is used for the treatment or prevention of HIV infection in a human subject who does not suffer from Kaposi""s sarcoma (KS). In the treatment of HIV infection, the Therapeutic of the invention can be used to prevent progression of HIV infection to ARC or to AIDS in a human patient, or to treat a human patient with ARC or AIDS.
In a preferred aspect of the invention, proteins, preferably xcex2-hCG peptides and derivatives are used to treat HIV infection. In particular, proteins, or nucleic acids encoding the proteins, containing an amino acid sequence of one or more portions of xcex2-hCG, preferably containing a sequence from amino acids 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145, 58-145, 109-145, 7-40, 46-65, or 48-56 (SEQ ID NOS:8-25 or 33-35, respectively) of FIG. 8 (a portion of SEQ ID NO:2), and preferably containing a sequence from amino acids 41-54, 45-54 or 109-119 (SEQ ID NOS:3, 4 or 7, respectively) of FIG. 8 (a portion of SEQ ID NO:2), and most preferably containing a sequence from amino acids 47-53 or 45-57 of FIG. 8 (a portion of SEQ ID NO:2), or circular [C44V]45-57 peptide (SEQ ID NO:26), or branched 45-57 (SEQ ID NO:6) peptide, or branched circular [V44C]45-57 peptide are used to treat HIV infection. In another embodiment, a protein is used which contains the amino acid sequence of two or more peptides of different, non-naturally contiguous portions of the xcex2-hCG sequence (FIG. 8 (SEQ ID NO:2)) in which the N-terminus of one portion is linked to the C-terminus of another portion by peptide bond(s). In a specific embodiment, a protein is used, the amino acid sequence of which consists of amino acids 45-57 (SEQ ID NO:6) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 109-119 (SEQ ID NO:7) or linked at the N-terminus via a peptide bond to the C-terminus of amino acids 110-119 (SEQ ID NO:27); or a protein is used that has an amino acid sequence of amino acids 47-57 (SEQ ID NO:28) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 108-119 (SEQ ID NO:29) of the xcex2-hCG sequence depicted in FIG. 8 (portions of SEQ ID NO:2); i.e., the fused peptides represented as 45-57::109-119, 110-119::45-57, or 47-57::108-119 (SEQ ID NOS:30-32, respectively). In yet another embodiment a protein is used which is a cyclic, fused peptide, particularly a cyclic, fused peptide having a sequence consisting of xcex2-hCG amino acids 110-119 (SEQ ID NO:27) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 45-57 (SEQ ID NO:6) and being circularized by a disulfide bond between the terminal cysteines at positions 110 and 57. In another embodiment, a protein is used that (a) comprises a xcex2-hCG amino acid sequence consisting of amino acid numbers 41-54, 45-54, 47-53, 45-57, 109-119, 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145, 58-145, 109-119, 109-145, 7-40, 46-65 or 48-56 (SEQ ID NOS:3-25 or 33-35, respectively) as depicted in FIG. 8 (a portion of SEQ ID NO:2) and (b) lacks xcex2-hCG amino acids contiguous to said sequence. In yet another embodiment, a purified derivative of a protein is used to treat or prevent HIV infection, the amino acid sequence of which protein is selected from the group consisting of amino acid numbers 41-54, 45-54, 47-53, 45-57, 109-119, 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145, 58-145, 109-145, 7-40, 46-65 or 48-56 (SEQ ID NOS:3-25 or 33-35, respectively) as depicted in FIG. 8 (a portion of SEQ ID NO:2). Other xcex2-hCG peptides, and nucleic acids encoding the peptides, and modifications and derivatives thereof, may have utility in the therapeutic methods of the invention.
In yet another embodiment, a fraction, particularly a size fraction, of a source of native hCG or native xcex2-hCG (i.e. naturally occurring, not recombinantly produced, hCG or xcex2-hCG) active in inhibiting HIV infection and replication, particularly a size fraction of approximately 40 kD, 15 kD or 2-3 kD, is used to treat or prevent HIV infection. The utility of xcex2-hCG peptides and fractions of native hCG and native xcex2-hCG sources may be determined by the in vitro and in vivo assays described herein or by any other method known in the art.
Additionally, the present inventors have found that different preparations of native hCG and native xcex2-hCG have variable effects on HIV infection both in vitro and in vivo. Specifically, the inventors found that among the commercial preparations of (non-recombinant) hCG they investigated, hCG from Fujisawa was the most effective, hCG APL(trademark) (Wyeth-Ayerst) the next most effective, and PREGNYL(trademark) (Organon) the next most effective in inhibiting HIV infection and replication. A highly purified hCG preparation and recombinant xcex2-hCG were found not to be active in inhibiting HIV infection in vitro. In fact, the present inventors have shown that specific size fractions of an active hCG preparation (APL(trademark); Wyeth Ayerst) have anti-HIV activity in vitro and anti-KS activity both in vitro and in vivo, as described herein.
These active fractions were eluted from the gel filtration sizing matrix as or close to (i.e., within 5 fractions (where the fractions are 4 ml fractions using a SUPERDEX(trademark) 200 column which is 26 mm2 by 60 mm)) the fractions containing or that would contain material that is approximately 40 kD (xc2x18 kD), 15 kD (xc2x13 kD) and 2-3 kD (xc2x12 kD) molecular weight. One skilled in the art would understand that these fractions could be subjected to further size fractionation to further isolate the component of these fractions having the anti-HIV and/or anti-KS activity. Additionally, other methods of fractionation, such as ion-exchange chromatography, affinity chromatography, etc., are well known in the art; those skilled in the art would be able to use any available fractionation techniques to obtain the active fractions from the active hCG preparations and human early (first trimester) pregnancy urine. hCG preparations and fractions of hCG preparations can be screened for efficacy in treating or preventing HIV infection, cancer, wasting syndrome, and/or hematopoietic deficiencies by the assays described herein or by any method known in the art. In a specific embodiment, the invention provides a first composition comprising one or more first components of a second composition comprising native hCG or native xcex2-hCG, said first components being separated from other components of the hCG or xcex2-hCG sample, said first components being active to inhibit HIV infection or replication or Kaposi""s sarcoma or having a pro-hematopoietic activity, and said second composition being active to inhibit HIV infection or replication or Kaposi""s sarcoma or having a pro-hematopoietic activity, and said native hCG or native xcex2-hCG not being purified to homogeneity in said second composition. In particular the invention provides a composition comprising components which have been separated from other components of the native hCG or native xcex2-hCG sample by sizing column chromatography, preferably where the components elute from a gel filtration, preferably a SUPERDEX(trademark) 200, sizing column with an apparent approximate molecular weight of 40 kD, 14 kD or 2-3 kD as determined relative to the elution of a native hCG heterodimer, having a molecular weight of 77 kD, and a xcex2-hCG core protein (xcex2-hCG amino acids 6-40 linked via a disulfide bond to xcex2-hCG amino acids 55-92, as depicted in FIG. 8 (SEQ ID NO:2)), having a molecular weight of 10 kD. In a specific embodiment, the therapeutic method of the invention is carried out as monotherapy, i.e., as the only agent provided for treatment or prevention of HIV. In another embodiment, the Therapeutic is administered in combination with one or more anti-viral compounds, for example, protease inhibitors (e.g., saquinavir) and/or reverse transcriptase inhibitors (e.g., azidothymidine (AZT), lamioridine (3TC), dideoxyinosine (ddI), dideoxycytidine (ddC)). The Therapeutic may also be administered in conjunction with chemotherapy (e.g., treatment with adriamycin, bleomycin, vincristine, vinblastine, doxorubicin and/or Taxol) or other therapies known in the art.
In another embodiment, HIV infection is treated or prevented by administration of a Therapeutic of the invention in combination with one or more chemokines. In particular, the Therapeutic is administered with one or more C-C type chemokines, especially one or more from the group RANTES, MIP-1xcex1 and MIP-1xcex2.
The invention provides for treatment or prevention of wasting syndrome by administration of a therapeutic compound (termed herein xe2x80x9cTherapeuticxe2x80x9d). Such xe2x80x9cTherapeuticsxe2x80x9d include, but are not limited to: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG (i.e. naturally occurring hCG or xcex2-hCG and not recombinantly produced hCG or xcex2-hCG), and therapeutically and prophylactically effective xcex2-hCG peptides, i.e., those fractions and peptides which prevent or treat wasting syndrome (e.g., as demonstrated in in vitro and in vivo assays described infra), and derivatives and analogs thereof, as well as nucleic acids encoding hCG, xcex2-hCG and therapeutically and prophylactically effective xcex2-hCG peptides and derivatives and analogs thereof (e.g., for use in gene therapy).
The methods of the invention can be used for treatment or prevention of any disease or disorder characterized by a loss of body cell mass. Particular conditions that can be treated by methods of the invention include, but are not limited to, wasting associated with viral, such as HIV, bacterial or other types of infections, and sepsis; cachexia associated with cancer, chemotherapy, and radiation therapy; wasting associated with chronic cardiovascular disease; wasting caused by exposure to toxic substances; wasting associated with diarrhea and other gastrointestinal disorders.
In a preferred embodiment, a Therapeutic of the invention is administered to treat or prevent a wasting syndrome associated with HIV infection. In another preferred embodiment, a Therapeutic of the invention is administered to treat or prevent a wasting syndrome associated with cancer.
Hematopoiesis
The invention also provides for treatment or prevention of diseases and disorders in which increased numbers of one or more hematopoietic cell types are desirable (e.g., diseases or disorders associated with one or more hematopoietic cell deficiencies) by administration of a therapeutic compound (termed herein xe2x80x9cTherapeuticxe2x80x9d) of the invention or by administration of hematopoietic cells, the production of which has been induced in vitro by contacting the cells with a Therapeutic of the invention. Such xe2x80x9cTherapeuticsxe2x80x9d include but are not limited to: hCG, xcex2-hCG and derivatives thereof, and therapeutically or prophylactically effective fractions of a source of native hCG or native xcex2-hCG and therapeutically or prophylactically effective xcex2-hCG peptides, i.e., those fraction and peptides which prevent or treat hematopoietic deficiencies (e.g., as demonstrated in in vitro and in vivo assays described infra) as well as modifications, derivatives and analogs thereof and nucleic acids encoding hCG, xcex2-hCG and therapeutically and prophylactically effective xcex2-hCG peptides, and derivatives and analogs thereof. In one embodiment, the Therapeutic of the invention is a protein containing an amino acid sequence of a therapeutically and prophylactically effective portion or portions of xcex2-hCG.
In a preferred embodiment, the Therapeutic of the invention is a protein having a sequence of amino acid numbers 41-54, 45-54, 47-53 or 45-57 (SEQ ID NOS:3-6, respectively) of the xcex2-hCG sequence depicted in FIG. 8 (a portion of SEQ ID NO:2). In other embodiments, the Therapeutic of the invention is a protein having a sequence of amino acid numbers 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145, 58-145, 109-145, 7-40, 46-65, or 48-56 (SEQ ID NOS:8-25 or 33-35, respectively) of the xcex2-hCG sequence of FIG. 8 (a portion of SEQ ID NO:2). Additionally, the present inventors have found that different preparations of hCG and xcex2-hCG have variable effects on hematopoietic cell proliferation in vitro and in vivo. Specifically, the inventors found that among the (non-recombinant) commercial preparations they investigated, hCG from Fujisawa was the most effective, hCG from APL (Wyeth-Ayerst) the next most effective, and pregnyl (Organon) the next most effective. hCG preparations and fractions of hCG and xcex2-hCG preparations can be screened for utility in inducing hematopoiesis in vitro or in vivo by the methods described infra herein or any method known in the art.
In one embodiment of the invention, the Therapeutic is administered directly to a patient suffering from a disease or disorder amenable to treatment by increasing production of one or more hematopoietic cell types (e.g., a disease or disorder associated with a hematopoietic cell deficiency). In another embodiment of the invention, hematopoietic cells, preferably stem and/or progenitor cells, are obtained, contacted with a Therapeutic of the invention in vitro to induce proliferation of the cells, and then administered to a subject suffering from a disease or disorder associated with a hematopoietic cell deficiency. Preferably, autologous hematopoietic cells (obtained from the subject or its identical twin) are reintroduced into the subject after in vitro expansion. In this embodiment, gene therapy methods can be performed by introducing a nucleic acid of interest, e.g., containing a gene which provides a function desired in a subject, into the hematopoietic cells, before or after expansion of the cells by contact with a Therapeutic. Hematopoietic cell subpopulations can be isolated for use, before or after expansion in vitro. For example, blood cells can be isolated and expanded, and optionally also differentiated, in vitro, followed by introduction of all or a portion of the cells (e.g, purified platelets, red blood cells, lymphocytes, etc.) into a patient.
In general, disorders that can be treated by methods of the invention include, but are not limited to, five broad categories. First are diseases resulting from a failure or dysfunction of normal blood cell production and maturation (i.e., aplastic anemia, cytopenias and hypoproliferative stem cell disorders). The second group are neoplastic, malignant diseases in the hematopoietic organs (e.g., leukemia and lymphomas). The third group of disorders comprises those of patients with a broad spectrum of malignant solid tumors of non-hematopoietic origin. Induction of hematopoietic cell proliferation or administration of replacement hematopoietic cells in these patients serves as a bone marrow rescue procedure, which is provided to a patient following otherwise lethal chemotherapy or irradiation of the malignant tumor. The fourth group of diseases consists of autoimmune conditions, where the hematopoietic cells serve as a source of replacement of an abnormal immune system. The fifth group of diseases comprises a number of genetic disorders which can be corrected by infusion of hematopoietic stem cells, preferably syngeneic, which prior to transplantation have undergone gene therapy. Particular diseases and disorders which can be treated by induction of hematopoietic cell production in vivo or by administration of hematopoietic cells expanded in vitro include but are not limited to those listed in Table 1, and described infra.
Treatment of Malignancies
Malignancies and related disorders that can be treated or prevented by administration of a Therapeutic of the invention include, but are not limited to, those disorders listed in Table 2 (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippencott Co., Philadelphia):
In specific embodiments, a Therapeutic of the invention is used to treat a neoplasm such as a gestational trophoblastic tumor, or testicular germ cell tumor, or cancer of the bladder, pancreas, cervix, lung, liver, ovary, colon or stomach, or adenocarcinoma or a virally induced cancer. In a preferred embodiment, a Therapeutic of the invention is used to treat neuroblastoma or carcinoma of the ovary or stomach. In a more preferred embodiment, a Therapeutic of the invention is used to treat Kaposi""s sarcoma or carcinoma of the breast, lung, prostate, or kidney (renal).
In one aspect of the invention, the Therapeutic is administered in conjunction with other cancer therapy, such as chemotherapy (e.g., treatment with adriamycin, bleomycin, vincristine, vinblastine, doxorubicin and/or Taxol).
The efficacy of a Therapeutic against a particular cancer can be determined by any method known in the art, for example but not limited to, those methods described herein.
The Therapeutics of the invention can also be administered to treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders listed in Table 2. Such prophylactic or therapeutic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79). Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
Alternatively or in addition to the presence of abnormal cell growth characterized as hyperplasia, metaplasia, or dysplasia, the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a patient, can indicate the desirability of prophylactic/therapeutic administration of a Therapeutic of the invention. Such characteristics of a transformed phenotype include morphology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, etc.
In other embodiments, a patient which exhibits one or more of the following predisposing factors for malignancy is treated by administration of an effective amount of a Therapeutic: a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome for chronic myelogenous leukemia, t(14;18) for follicular lymphoma, etc.), familial polyposis or Gardner""s syndrome (possible forerunners of colon cancer), benign monoclonal gammopathy (a possible forerunner of multiple myeloma), and a first degree kinship with persons having a cancer or precancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the colon, Gardner""s syndrome, hereditary exostosis, polyendocrine adenomatosis, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen, retinoblastoma, carotid body tumor, cutaneous melanocarcinoma, intraocular melanocarcinoma, xeroderma pigmentosum, ataxia telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi""s aplastic anemia, and Bloom""s syndrome; see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 112-113, etc.)
In another specific embodiment, a Therapeutic of the invention is administered to a human patient to prevent progression to breast, colon, lung, pancreatic, or uterine cancer, or melanoma or sarcoma.
Hyperproliferative malignant stem cell disorders as well as non-hematopoietic malignancies can be treated with chemotherapy or radiation therapy along with rescue of hematopoietic cells by direct administration of a Therapeutic of the invention or by administration of hematopoietic cells induced to proliferate by contacting the cells with a Therapeutic of the invention. The conditions that can be treated according to the invention include, but are not limited to, the leukemias listed in Table 2 and the solid tumors listed in Table 2.
These malignancies are currently treated by, inter alia, chemotherapy and, when feasible, allogeneic bone marrow transplantation. However, allogeneic HLA identical sibling bone marrow is available only to less than one-third of patients, and this treatment is associated with transplantation-related complications such as immunodeficiency and graft versus host disease. Induction of hematopoietic cell proliferation in vivo or provision of autologous hematopoietic stem and progenitor cells expanded by administration of a Therapeutic in vitro permits hematopoietic reconstitution of patients lacking suitable allogeneic donors and eliminates the risks of graft versus host disease arising from allogeneic marrow transplantation. Thus, in a specific embodiment, a Therapeutic is used to induce proliferation in hematopoietic cells which are then administered to a patient who has undergone chemotherapy or radiation therapy for treatment of cancer or an immunological disorder. In another embodiment, a Therapeutic is directly administered to a patient who has undergone chemotherapy or radiation therapy for treatment of cancer or an immunological disorder.
Autoimmune Disorders
Many chronic inflammatory and degenerative diseases are characterized by a continuous immune reaction against the body""s own tissues. Such autoimmune disorders include but are not limited to rheumatoid arthritis and other inflammatory osteopathies, diabetes type I, chronic hepatitis, multiple sclerosis, and systemic lupus erythematosus. Autoimmune disorders are often treated by lymphoid irradiation. Administration of a Therapeutic of the invention or of cells produced by exposure to a Therapeutic in vitro can be valuable to repopulate the hematopoietic system after radiotherapy.
Anti-inflammatory drugs such as steroids retard the inflammatory cells which are activated by autoreactive T cells, but do not prevent T cells which recognize self-proteins from activating new inflammatory cells. A more direct approach to treating autoimmune diseases depends on eradication of T cells by irradiation of the lymphoid tissues, and relying on stem cells from the unirradiated bone marrow to repopulate the patient""s hematopoietic system. The rationale is that the formation of new populations of mature T cells from bone marrow stem cells may result in absence of T cells that have reactivity to self-specific antigens. This procedure, called total lymphoid irradiation (TLI), has been used to treat intractable rheumatoid arthritis (Strober, S., et al., 1985, Annals of Internal Medicine 102:441-449, 450-458). These clinical trials showed that in the majority of otherwise intractable cases, joint disease was significantly alleviated for at least 2-3 years. However, the major drawback to such treatment is failure of stem cells in the bone marrow of these elderly patients to efficiently repopulate the hematopoietic system, resulting in infections and bleeding disorders. Analogous studies have been made of the effects of TLI as an alternative to cytotoxic drugs for treatment of SLE (Strober, S., et al., 1985, Ann. Internal Med. 102:450). Studies of the use of TLI to treat intractable SLE have also shown that this treatment alleviates disease activity, but is severely limited by failure of bone marrow stem cells to rapidly and efficiently repopulate the hematopoietic system after irradiation.
Thus, a Therapeutic of the invention can be administered to promote proliferation of the remaining hematopoietic cells to increase the success of TLI therapy. Additionally, hematopoietic stem and progenitor cells can be isolated from the patient before treatment, induced to proliferate in vitro and then introduced into the patient after TLI treatment to repopulate the hematopoietic system.
Diseases Resulting from a Failure or Dysfunction of Normal Blood Cell Production and Maturation
In a preferred aspect, a Therapeutic of the invention is used to treat a disease resulting from a failure or dysfunction of normal blood cell production and maturation, such as an aplastic anemia, a cytopenia or a hypoproliferative stem cell disorder. These disorders entail failure of stem cells in bone marrow to provide normal numbers of functional blood cells. The aplastic anemias result from the failure of stem cells to give rise to the intermediate and mature forms of red cells, white cells, and platelets. While red cell production is usually most seriously affected, a marked decrease in production of other mature blood cell elements is also seen as some anemias specifically affect production of white cells and/or platelets. The large majority of these anemias are acquired during adult life, and do not have any apparent genetic predisposition. About half of these acquired anemias arise in the absence of any obvious causative factor such as exposure to poisons, drugs or disease processes that impair stem cell function; these are termed idiopathic aplastic anemias. The remaining cases are associated with exposure to an extremely diverse array of chemicals and drugs and also occur as the consequence of viral infections, such as HIV infection, and after pregnancy. Other specific types of aplastic anemia are termed agranulocytosis or thrombocytopenia to indicate that the major deficiency lies in particular white cells or in platelet production, respectively. These non red blood cell deficiencies are also often associated with HIV infection. Also significantly associated with HIV infection is a severe platelet deficiency, Idiopathic Thrombocytopenic Purpura (ITP). Additionally, agranulocytosis may be associated with autoimmune syndromes such as systemic lupus erythematosus (SLE) or with other infections, such as neonatal rubella.
In addition, immune deficiencies which are the primary or secondary result of infection by pathogenic microorganisms can be treated by administration of a Therapeutic of the invention. For example, immune deficiencies caused by microorganisms which are intracellular pathogens of hematopoietic cells, can be treated by the provision of new hematopoietic cells. These new hematopoietic cells can be generated by contacting hematopoietic stem and/or progenitor cells in vitro with a Therapeutic of the invention to cause proliferation of the cells. Microorganisms causing immune deficiencies which may be treated according to this embodiment of the invention include but are not limited to gram-negative bacilli such as Brucella or Listeria, the mycobacterium which are the etiological agents of tuberculosis or of Hansen""s disease (leprosy), parasites such as Plasmodium (the etiological agents of malaria) or Leishmania, and fungi (such as those that cause pneumonia and other lethal infections secondary to immunodeficiencies) (for a discussion of many of these disorders, see Harrison""s Principles of Internal Medicine, 1970, 6th Edition, Wintrobe, M. M., et al., eds., McGraw-Hill, New York, pp. 798-1044).
In a preferred embodiment of the invention, a Therapeutic of the invention is administered for the treatment of a cytopenia associated with HIV infection.
The hematopoietic deficiencies associated with HIV infection include reduction in CD4+ T cells and other lymphocytes, red blood cells, platelets, specifically ITP, and neutrophils. Such a disorder is treated by contacting hematopoietic stem and/or progenitor cells in vitro with a Therapeutic of the invention and then infusing the resulting hematopoietic cells into the subject in need of treatment. In a another preferred embodiment, the disorder is treated by direct administration of a Therapeutic of the invention to the subject in need of treatment. Assays for determining the efficacy of particular Therapeutics for treatment of hematopoietic deficiencies associated with HIV infection are detailed herein.
Sources of hCG and xcex2-hCG
Native preparations (i.e. derived from naturally occurring sources and not recombinantly produced) of hCG and xcex2-hCG can be obtained from a variety of sources. Both hCG and xcex2-hCG are commercially available (e.g., Sigma Chemical Company) and hCG is commercially available in a form suitable for therapeutic use in humans (e.g., from Fujisawa, Wyeth-Ayerst Laboratories (APL(trademark)), Organon, Inc. (PREGNYL(trademark)) and Serono Laboratories, Inc. (PROFASI(trademark))). hCG is also present at particularly high concentrations in the urine of women in the first trimester of pregnancy (xe2x80x9chuman early pregnancy urinexe2x80x9d). Other sources include, but are not limited to, urine from women in the second and third trimesters of pregnancy, urine from patients with proteinuria, urine from patients having hCG secreting tumors or other cancer patients, and from pituitary glands.
Since the inventors have discovered that different sources of hCG have variable effects on HIV infection and cancer cell growth in vitro and in vivo, one aspect of the invention relates to assaying preparations of hCG for efficacy in treatment or prevention of HIV infection. The therapeutic effectiveness of hCG preparations and fractions can be tested by the in vitro or in vivo assays described herein or by any method known in the art. It is preferable to test the hCG preparation or fraction in an in vitro assay, e.g., for HIV replication or transcription from the HIV-1 LTR or in vivo in an animal model, such as HIV transgenic mice or SIV infected monkeys, before assaying the preparation in humans.
In a specific embodiment, a preparation comprising hCG is used that contains not only the hCG heterodimer but also peptide fragments thereof, e.g., xcex2 chain peptides.
hCG and xcex2-hCG can also be purified, or preferably partially purified, from any source known to contain hCG or xcex2-hCG, e.g., urine from pregnant women, using conventional techniques well-known in the art, such as affinity chromatography. For example, antibodies prepared against hCG or xcex2-hCG can be used to prepare an affinity chromatography column which can be used to purify the proteins by well-known techniques (see, e.g., Hudson and May, 1986, Practical Immunology, Blackwell Scientific Publications, Oxford, United Kingdom).
The xcex2-hCG-related proteins are preferably prepared by any chemical or enzymatic synthesis method known in the art, as described supra herein.
Fractionation of Sources of hCG
The present inventors have found that the component(s) of a source of hCG having therapeutic activity can be further isolated by fractionation of the source of hCG. The inventors have fractionated the active portions of the commercial hCG preparation APL(trademark) (Wyeth-Ayerst) and human early pregnancy urine as described herein. Other sources of hCG include, but are not limited to, urine from women in the second and third trimester of pregnancy, urine from proteinuria patients (both pregnant women with preeclampsia and patients with nephrotic syndromes), urine from patients with hCG secreting tumors, and pituitary glands. However, those skilled in the art will appreciate that any source of hCG or xcex2-hCG having anti-HIV activity and/or anti-KS activity and/or a pro-hematopoietic effect can be fractionated to further isolate the active components. The source of hCG or xcex2-hCG can be fractionated using any technique available in the art for the separation and isolation of molecules, for example but not limited to, sizing chromatography, ion-exchange chromatography, affinity chromatography, etc.
Additionally, the present inventors have found that different preparations of hCG and xcex2-hCG have variable effects on HIV infection, KS cell growth and hematopoiesis both in vitro and in vivo. Specifically, the inventors found that among the commercial preparations of (non-recombinant) hCG they investigated, hCG from Fujisawa was the most effective, hCG APL(trademark) (Wyeth-Ayerst) the next most effective, and PREGNYL(trademark) (Organon) the next most effective in inhibiting HIV infection and replication. A highly purified hCG preparation and recombinant xcex2-hCG were found not to be active in inhibiting HIV infection, KS cell growth and promoting hematopoiesis in vitro. In fact, the present inventors have shown that specific size fractions of an active hCG preparation (APL(trademark); Wyeth Ayerst) and human early (i.e. first trimester) pregnancy urine have anti-HIV and pro-hematopoieitic activities in vitro and anti-KS activity both in vitro and in vivo, as described infra herein.
These active fractions were eluted from the sizing matrix as or close to (i.e., within 5 fractions (where the fractions are 4 ml fractions using a SUPERDEX(trademark) 200 (Pharmacia) column which is 26 mm2 by 60 mm)) the fractions containing or that would contain material that is approximately 40 kD (xc2x18 kD), 15 kD (xc2x13 kD) and 2-3 kD (xc2x12 kD) molecular weight. One skilled in the art would understand that these fractions could be subjected to further size fractionation to further isolate the component of these fractions having the anti-HIV and/or anti-KS activity. Additionally, other methods of fractionation, such as ion-exchange chromatography, affinity chromatography are well known in the art; those skilled in the art would be able to use any available fractionation techniques to obtain the active fractions from the active hCG preparations and human early (first trimester) pregnancy urine (or any other preparation containing hCG or xcex2-hCG).
In a specific embodiment, the invention provides a first composition comprising one or more first components of a second composition comprising native hCG or native xcex2-hCG, said first components being separated from other components of the hCG or xcex2-hCG sample, said first components being active to inhibit HIV infection or replication or Kaposi""s sarcoma or having a pro-hematopoietic activity, and said second composition being active to inhibit HIV infection or replication or Kaposi""s sarcoma or having a pro-hematopoietic activity, and said native hCG or native xcex2-hCG not being purified to homogeneity in said second composition.
In particular the invention provides a composition comprising components which have been separated from other components of the native hCG or native xcex2-hCG sample by sizing column chromatography, preferably where the components elute from a gel filtration, preferably a SUPERDEX(trademark) 200, sizing column with an apparent approximate molecular weight of 40 kD, 14 kD or 2-3 kD as determined relative to the elution of a hCG heterodimer, having a molecular weight of 77 kD, and a xcex2-hCG core protein (xcex2-hCG amino acids 6-40 linked via a disulfide bond to xcex2-hCG amino acids 55-92, as depicted in FIG. 8 (SEQ ID NO:2)), having a molecular weight of 10 kD. hCG preparations and fractions of hCG preparations can be screened for efficacy in inducing proliferation of hematopoietic cells by the assays described herein or by any method known in the art.
Thus, in a preferred embodiment, a fraction, particularly a size fraction, of a source of native hCG or native xcex2-hCG active in promoting hematopoiesis, particularly a size fraction of approximately 40 kD, 15 kD or 2-3 kD, is used to treat or prevent a hematopoietic deficiency.
The inventors have fractionated the active portions of the commercial hCG preparation APL(trademark) (Wyeth-Ayerst) and human early (i.e. first trimester) pregnancy urine as described herein. Other sources of hCG include, but are not limited to, urine from women in the second and third trimester of pregnancy, urine from proteinuria patients (both pregnant women with preeclampsia and patients with nephrotic syndromes), urine from patients with hCG secreting tumors, and pituitary glands. However, those skilled in the art will appreciate that any source of hCG or xcex2-hCG having anti-HIV activity and/or anti-KS activity and/or a pro-hematopoietic effect can be fractionated to further isolate the active components. The source of hCG or xcex2-hCG can be fractionated using any technique available in the art for the separation and isolation of molecules, for example but not limited to, sizing chromatography, ion-exchange chromatography, affinity chromatography, etc.
Briefly, by way of example but not by way of limitation, urine can be prepared for fractionation as follows:
Urine is collected and stored either frozen or refrigerated for not more than two (2) days. Then, sodium azide is then added at a concentration of 1 gram/liter and the sample is stored frozen until sufficient material is collected for the fractionation.
At this point, the urine is thawed over night, the pH adjusted to 7.2 to 7.4 with sodium hydroxide and then centrifuged to remove any precipitate (alternatively, the precipitate can be allowed to sediment, e.g., for 1 hour at room temperature, approximately 75% of the supernatant is decanted, the remainder of the supernatant and the precipitate is centrifuged to pellet the precipitate, and the supernatant decanted and added to the first volume of decanted supernatant). The urine is then filtered through, e.g., a 45 micron filter to remove any remaining particulate matter.
Next, the urine is concentrated using any concentration method available in the art which does not remove higher molecular weight material, e.g., material larger than 3,000 daltons in molecular weight; for example, the material may be concentrated using a Pellicon (Millipore) filtration system with a membrane filter cassette having a molecular weight cut off of 3,000 daltons. Concentration with the Pellicon filtration system using the 3,000 molecular weight membrane filter cut off concentrates 30 liters of urine to 500 ml (i.e., a 60-fold concentration) overnight.
To remove salts and lipids, the concentrate can then be passed over a column containing a large volume of Sephadex G25 resin in 0.05 M ammonium bicarbonate (for example, 250 ml of the concentrate can be passed over a column of approximately 1.7 liters, washing the column with 25% ethanol between runs to remove adsorbed lipids and glycoprotein). The resulting desalted and delipidated urine concentrate is then lyophilized.
The lyophilized urine material or commercial hCG preparation (or any source of native hCG or xcex2-hCG) is resuspended in either phosphate buffered saline (PBSxe2x80x9430 mM sodium phosphate buffer, pH 8.3) or in 0.10 M ammonium bicarbonate at a concentration and in a volume appropriate for the column upon which the sample will be loaded, for example, but not limited to 0.5 grams of protein in 6 ml (i.e., approximately 83 mg/ml). It is within the skill of the skilled artisan to determine the concentration and volume of the sample to be subjected to fractionation.
The sample can then be fractionated by any method known in the art for the separation of proteins. A preferred method is high resolution gel filtration on a Pharmacia pre-packed SUPERDEX(trademark) 200 column (26/60) by HPLC using any available HPLC apparatus, e.g., with a Hewlett Packard 1050 HPLC equipped with a photodiode array detector. The resuspended sample is loaded onto the column in 30 mM phosphate buffer, pH 8.3, and the material can then be eluted from the column with 30 mM sodium phosphate buffer, pH 7.0; 2M NaCl in e.g. 4 ml fractions. Fractionation can also be performed using other types of chromatography matrices e.g., heparin, DEAE-cellulose, Sephadex A50, Sephadex G100, phenyl sepharose, or any sizing, ion-exchange, affinity chromatography or any other chromatography matrix available in the art. The column chromatography can also be run using any method available in the art, e.g., standard gravity flow or low pressure chromatography, FPLC, or reverse phase HPLC.
Many separation techniques are known in the art. Those skilled in the art would understand how to apply these known techniques to the fractionation of hCG preparations.
Once the material has been fractionated, any method known in the art, such as but not limited to, those described herein, can be used to determine which fractions have anti-HIV activity and/or anti-KS activity and/or a pro-hematopoietic effect.
When fractionating by size, such as fractionation on the SUPERDEX(trademark) 200 column, the apparent molecular weight of material in the fractions can be determined by the relative elution of those fractions compared with the elution of specific hCG and xcex2-hCG species having a known molecular weight or with the elution of known protein size markers. In general, proteins elute from a sizing column as a function of their molecular weight. The elution of, for example, hCG and the xcex2-hCG core protein can be determined by assaying the column chromatography fractions for the presence of hCG and the xcex2-hCG core protein, or any hCG or xcex2-hCG species, by any immunoassay technique available in the art, such as radioimmunoassays (either liquid or solid phase), enzyme-linked assays or ELISA assays.
Antibodies, either polyclonal or, preferably, monoclonal, can be generated against hCG or the xcex2-hCG core protein by any method known in the art. Preparation of monoclonal antibodies against hCG and xcex2-hCG species have been described in the art, see, e.g., O""Connor et al., 1994, Endocrine Reviews 15:650-683; Krichevsky et al, 1991, Endocrinology 128:1255-1264; and Krichevsky et al., 1988, Endorcrinology 123:584-593. For the production of antibodies, various host animals can be immunized by injection with hCG, the xcex2-hCG core protein or any other species of hCG, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund""s (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. For preparation of monoclonal antibodies, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Monoclonal cells lines can then be screened for binding to the particular hCG or xcex2-hCG species using the purified species in any type of immunoassay available in the art (see, e.g., Erlich et al., 1985, Am. J. Reprod Immunol. Microbiol. 8:48).
The fractions can then be assayed for the presence of the hCG or xcex2-hCG species using a monoclonal antibody specific for the hCG or xcex2-hCG species. The assay can be performed by any method known in the art. For example, an immunoradiometric assay (IRMA) can be used (Krichevsky et al., 1988, Endocrinology 123:584-593). Briefly, the IRMA assay is performed by adsorbing an antibody against the hCG or xcex2-hCG species onto the surface of wells of a microtiter plate by incubation in a coating buffer (0.2 M sodium bicarbonate, pH 9.5) overnight at 4xc2x0 C. The residual non-specific binding sites are blocked by the addition of a 1% bovine serum albumin solution (with 0.1% sodium azide) to the wells for 3 hours at room temperature, and the wells of the microtiter plate are then washed with deionized water. An aliquot of the fraction in assay buffer (0.01 M sodium phosphate, 0.15 M NaCl, 0.01 M EDTA, 0.1% sodium azide, 0.1% bovine xcex3-globulin, pH 7.4) is incubated in the wells for 24 hours at room temperature. The sample is then removed and the wells washed with deionized water. A solution of a second antibody specific for the hCG or xcex2-hCG species, which antibody has been iodinated with I125, (approximately 40,000 cpm/well) is incubated in the wells for 24 hours at room temperature. The iodinated antibody solution is removed and the wells washed five times with deionized water. The level of radioactivity in each well is then determined in a scintillation counter which can measure xcex3-irradiation.
In a specific embodiment, a preparation comprising hCG is used that contains not only the hCG heterodimer but also peptide fragments thereof, e.g., xcex2 chain peptides.
In a preferred embodiment of the invention, proteins (e.g., peptides), the amino acid sequence of which consists of one or more portions effective to increase the production of one or more hematopoietic cell types of the xcex2-hCG sequence (xcex2-hCG peptides) are used to treat or prevent HIV infection, cancer, wasting syndrome and/or hematopoietic deficiencies. In various specific embodiments, the portion(s) of the xcex2-hCG sequence are at least 3, 5, 10, 20, or 30 amino acids. These proteins are preferably xcex2-hCG peptides, or nucleic acids encoding xcex2-hCG peptides, from amino acids 41-54, 45-54, 47-53 and 45-57 (SEQ ID NOS:3-6, respectively) of FIG. 8 (a portion of SEQ ID NO:2). In other embodiments, these proteins are xcex2-hCG peptides, or nucleic acids encoding xcex2-hCG peptides, of amino acids 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145, 58-145, 109-145, 7-40, 46-65, or 48-56 (SEQ ID NOS:8-25 or 33-35, respectively) of FIG. 8 (a portion of SEQ ID NO:2), and preferably containing a sequence from amino acids 41-54, 45-54 or 109-119 (SEQ ID NOS:3, 4 or 7, respectively) of FIG. 8 (a portion of SEQ ID NO:2), and most preferably containing a sequence from amino acids 47-53 or 45-57 of FIG. 8 (a portion of SEQ ID NO:2), or circular [C44V]45-57 peptide (SEQ ID NO:26), or branched 45-57 (SEQ ID NO:6) peptide, or branched circular [V44C]45-57 peptide are used to treat or prevent hematopoietic deficiencies.
In another embodiment, the invention provides proteins, the amino acid sequences of which consist of two or more at least 5, 7 or 10 amino acid, non-naturally contiguous portions of the xcex2-hCG sequence (FIG. 8 (SEQ ID NO:2)) linked by peptide bonds between the N-terminus of one portion and the C-terminus of another portion. Specifically, proteins, the amino acid sequences of which consist of amino acids 45-57 (SEQ ID NO:6) linked at the C-terminus via a peptide bond to the N-terminus of a peptide of amino acids 109-119 (SEQ ID NO:7) or linked at the N-terminus via a peptide bond to the C-terminus of amino acids 110-119 (SEQ ID NO:27); or an isolated protein of amino acids 47-57 (SEQ ID NO:28) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 108-119 (SEQ ID NO:29) of the xcex2-hCG sequence depicted in FIG. 4 (portions of SEQ ID NO:2), i.e., the fused peptides denoted as 45-57::109-119, 110-119::45-57, or 47-57::108-119 (SEQ ID NOS:30-32, respectively). Derivatives of the foregoing fusion proteins are also provided (e.g., branched, cyclized, N- or C-terminal chemically modified, etc.). In another embodiment, fusion proteins comprising two or more such portions of the xcex2-hCG sequence are provided; such portions may or may not be contiguous to one another (i.e., an intervening sequence may be present). Molecules comprising such portions linked by hydrocarbon linkages are also provided. In another embodiment, the peptides of the invention (i) have an amino acid sequence consisting of no more than 8 peptides of the xcex2-hCG sequence (FIG. 8 (SEQ ID NO:2)) and (ii) comprise amino acid numbers 47-53 (SEQ ID NO:5) of xcex2-hCG (FIG. 8 (SEQ ID NO:2)).
In another embodiment, a protein is used that (a) comprises one or more portions of the amino acid sequence consisting of xcex2-hCG, a peptide having an amino acid sequence consisting of said portion(s) being effective to increase production of one or more hematopoietic cell types; and (b) lacks xcex2-hCG amino acids contiguous to said portion(s). In another embodiment, a protein is used that (a) comprises a xcex2-hCG amino acid sequence consisting of amino acid numbers 41-54, 45-54, 47-53, 45-57, 109-119, 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145, 58-145, 109-119, 109-145, 7-40, 46-65 or 48-56 (SEQ ID NOS:3-25 or 33-35, respectively) as depicted in FIG. 8 (a portion of SEQ ID NO:2) and (b) lacks xcex2-hCG amino acids contiguous to said sequence. Peptides containing the above sequences in which only conservative substitutions have been made are also provided by the present invention, as but one example of peptide derivatives within the scope of the invention. Analogs of the above-mentioned proteins and peptides which have one or more amino acid substitutions forming a branched peptide (e.g., by substitution with an amino acid or amino acid analog having a free amino- or carboxy-side chain that forms a peptide bond with a sequence of one or more amino acids, including but not limited to prolines) or allowing circularization of the peptide (e.g., by substitution with a cysteine, or insertion of a cysteine at the amino- or carboxy-terminus or internally), to provide a sulfhydryl group for disulfide bond formation, are also provided.
Other xcex2-hCG peptides, and nucleic acids encoding these peptides, may have utility in the therapeutic methods of the invention. The utility of xcex2-hCG peptides may be determined by the in vitro and in vivo assays described in herein or by any other method known in the art.
In specific embodiments, peptides of less than 50, or less than 25, amino acids are provided.
The invention also relates to derivatives, modifications and analogs of xcex2-hCG peptides. One embodiment of the invention provides a purified derivative of a protein effective to increase the production of one or more hematopoietic cell types, which protein contains an amino acid sequence of one or more portions effective to increase the production of one or more hematopoietic cell types of xcex2-hCG. Another embodiment of the invention provides a purified derivative of a protein effective to increase the production of one or more hematopoietic cell types, the amino acid sequence of which protein is selected from the group consisting of amino acid numbers 41-54, 45-54, 47-53, 45-57, 109-119, 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145, 58-145, 109-145, 7-40, 46-65 or 48-56 (SEQ ID NOS:3-25 or 33-35, respectively) as depicted in FIG. 8 (a portion of SEQ ID NO:2). In another embodiment, xcex2-hCG peptide derivatives can be made by altering the xcex2-hCG peptide sequence by substitutions, additions or deletions that provide for therapeutically effective molecules. Thus, the xcex2-hCG peptide derivatives include peptides containing, as a primary amino acid sequence, all or part of the particular xcex2-hCG peptide sequence including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a peptide which is functionally active. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Conservative substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such xcex2-hCG peptide derivatives can be made either by chemical peptide synthesis or by recombinant production from a nucleic acid encoding the xcex2-hCG peptide which nucleic acid has been mutated. Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem 253:6551), use of TAB(copyright) linkers (Pharmacia), etc.
In addition, xcex2-hCG peptides and analogs and derivatives of xcex2-hCG peptides can be chemically synthesized. (See, e.g., Merrifield, 1963, 1. Amer. Chem. Soc. 85:2149-2156.) For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., see Creighton, 1983, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., N.Y., pp. 50-60). xcex2-hCG peptides can also be synthesized by use of a peptide synthesizer. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., N.Y., pp. 34-49). Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the xcex2-hCG peptide. Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, xcex1-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, xcex3-Abu, xcex5-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, xcex2-alanine, fluoro-amino acids, designer amino acids such as xcex2-methyl amino acids, Cxcex1-methyl amino acids, Nxcex1-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).
By way of example but not by way of limitation, peptides of the invention can be chemically synthesized and purified as follows: Peptides can be synthesized by employing the N-xcex1-9-fluorenylmethyloxycarbonyl or Fmoc solid phase peptide synthesis chemistry using a Rainin Symphony Multiplex Peptide Synthesizer. The standard cycle used for coupling of an amino acid to the peptide-resin growing chain generally includes: (1) washing the peptide-resin three times for 30 seconds with N,N-dimethylformamide (DMF); (2) removing the Fmoc protective group on the amino terminus by deprotection with 20% piperdine in DMF by two washes for 15 minutes each, during which process mixing is effected by bubbling nitrogen through the reaction vessel for one second every 10 seconds to prevent peptide-resin settling; (3) washing the peptide-resin three times for 30 seconds with DMF; (4) coupling the amino acid to the peptide resin by addition of equal volumes of a 250 mM solution of the Fmoc derivative of the appropriate amino acid and an activator mix consisting or 400 mM N-methylmorpholine and 250 mM (2-(1H-benzotriazol-1-4))-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) in DMF; (5) allowing the solution to mix for 45 minutes; and (6) washing the peptide-resin three times for 30 seconds of DMF. This cycle can be repeated as necessary with the appropriate amino acids in sequence to produce the desired peptide. Exceptions to this cycle program are amino acid couplings predicted to be difficult by nature of their hydrophobicity or predicted inclusion within a helical formation during synthesis. For these situations, the above cycle can be modified by repeating step 4 a second time immediately upon completion of the first 45 minute coupling step to xe2x80x9cdouble couplexe2x80x9d the amino acid of interest. Additionally, in the first coupling step in peptide synthesis, the resin can be allowed to swell for more efficient coupling by increasing the time of mixing in the initial DMF washes to three 15 minute washes rather than three 30 second washes. After peptide synthesis, the peptide can be cleaved from the resin as follows: (1) washing the peptide-resin three times for 30 seconds with DMF; (2) removing the Fmoc protective group on the amino terminus by washing two times for 15 minutes in 20% piperdine in DMF; (3) washing the peptide-resin three times for 30 seconds with DMF; and (4) mixing a cleavage cocktail consisting of 95% trifluoroacetic acid (TFA), 2.4% water, 2.4% phenol, and 0.2% triisopropysilane with the peptide-resin for two hours, then filtering the peptide in the cleavage cocktail away from the resin, and precipitating the peptide out of solution by addition of two volumes of ethyl ether. Specifically, to isolate the peptide, the ether-peptide solution can be allowed to sit at xe2x88x9220xc2x0 C. for 20 minutes, then centrifuged at 6,000xc3x97G for 5 minutes to pellet the peptide, and the peptide can be washed three times with ethyl ether to remove residual cleavage cocktail ingredients. The final peptide product can be purified by reversed phase high pressure liquid chromatography (RP-HPLC) with the primary solvent consisting of 0.1% TFA and the eluting buffer consisting of 80% acetonitrile and 0.1% TFA. The purified peptide can then be lyophilized to a powder.
In a preferred embodiment, the invention provides a peptide with branched amino acids (branched peptide), preferably a branched peptide of amino acids 45-57 (SEQ ID NO:6) with branches occurring at positions 47 and 51, respectively, instead of the Gly and Ala residues normally present. Most preferably, diaminobutyric acid is substituted for the gly and ala residues at positions 47 and 51, respectively, and proline bonded to both diaminobutyric acid residues (45-57 branched) as shown in FIG. 9A.
In other specific embodiments, branched versions of the xcex2-hCG peptides listed hereinabove are provided, e.g., by substituting one or more amino acids within the xcex2-hCG sequence with an amino acid or amino acid analog with a free side chain capable of forming a peptide bond with one or more amino acids (and thus capable of forming a xe2x80x9cbranchxe2x80x9d).
Branched peptides may be prepared by any method known in the art for covalently linking any naturally occurring or synthetic amino acid to any naturally occurring or synthetic amino acid in a peptide chain which has a side chain group able to react with the amino or carboxyl group on the amino acids so as to become covalently attached to the peptide chain. In particular, amino acids with a free amino side chain group, such as, but not limited to, diaminobutyric acid, lysine, arginine, ornithine, diaminopropionic acid and citrulline, can be incorporated into a peptide so that an amino acid can form a branch therewith, for example, by forming a peptide bond to the free amino side group, from that residue. Alternatively, amino acids with a free carboxyl side chain group, such as, but not limited to, glutamic acid, aspartic acid and homocitrulline, can be incorporated into the peptide so that an amino acid can form a branch therewith, for example, by forming a peptide bond to the free carboxyl side group, from that residue. The amino acid forming the branch can be linked to a side chain group of an amino acid in the peptide chain by any type of covalent bond, including, but not limited to, peptide bonds, ester bonds and disulfide bonds. In a specific embodiment, amino acids, such as those described above, that are capable of forming a branch point, are substituted for xcex2-hCG residues within a peptide having a xcex2-hCG sequence.
Branched peptides can be prepared by any method known in the art. For example, but not by way of limitation, branched peptides can be prepared as follows: (1) the amino acid to be branched from the main peptide chain can be purchased as an N-xcex1-tert-butyloxycarbonyl (Boc) protected amino acid pentafluorophenyl (Opfp) ester and the residue within the main chain to which this branched amino acid will be attached can be an N-Fmoc-xcex1-xcex3-diaminobutyric acid; (2) the coupling of the Boc protected amino acid to diaminobutyric acid can be achieved by adding 5 grams of each precursor to a flask containing 150 ml DMF, along with 2.25 ml pyridine and 50 mg dimethylaminopyridine and allowing the solution to mix for 24 hours; (3) the peptide can then be extracted from the 150 ml coupling reaction by mixing the reaction with 400 ml dichlormethane (DCM) and 200 ml 0.12N HCl in a 1 liter separatory funnel, and allowing the phases to separate, saving the bottom aqueous layer and re-extracting the top layer two more times with 200 ml 0.12 N HCl; (4) the solution containing the peptide can be dehydrated by adding 2-5 grams magnesium sulfate, filtering out the magnesium sulfate, and evaporating the remaining solution to a volume of about 2-5 ml; (5) the dipeptide can then be precipitated by addition of ethyl acetate and then 2 volumes of hexanes and then collected by filtration and washed two times with cold hexanes; and (6) the resulting filtrate can be lyophilized to achieve a light powder form of the desired dipeptide. Branched peptides prepared by this method will have a substitution of diaminobutyric acid at the amino acid position which is branched. Branched peptides containing an amino acid or amino acid analog substitution other than diaminobutyric acid can be prepared analogously to the procedure described above, using the N-F-moc coupled form of the amino acid or amino acid analog.
In a preferred embodiment, the peptide is a cyclic peptide, preferably a cyclic peptide of xcex2-hCG amino acids 44-57 (SEQ ID NO:26) with cysteine substituted for valine at position 44 and circularized via a disulfide bond between the cysteine residues at positions 44 and 57 (C[V44C] 45-57) (FIG. 9B) or a cyclic fused peptide of xcex2-hCG amino acids 110-119 (SEQ ID NO:27) linked at the C-terminus by a peptide bond to the N-terminus of amino acids 45-57 (SEQ ID NO:6) and circularized via a disulfide bond between the cysteine residues at positions 110 and 57. In another preferred embodiment, the peptide is a cyclic branched peptide of xcex2-hCG amino acids 44-57 (SEQ ID NO:12) with cysteine substituted for valine at position 44 and circularized via a disulfide bond between the cysteine residues at positions 44 and 57 and positions 47 and 51 substituted with a diaminobutyric acid residue on which a proline is peptide bonded to its free amino sidechain.
Cyclization can be, for example, but not by way of limitation, via a disulfide bond between two cysteine residues or via an amide linkage. For example, but not by way of limitation, disulfide bridge formation can be achieved by (1) dissolving the purified peptide at a concentration of between 0.1.-0.5 mg/ml in 0.01 M ammonium acetate, pH 7.5; (2) adding to the dissolved peptide 0.01 M potassium ferricyanide dropwise until the solution appears pale yellow in color and allowing this solution to mix for 24 hours; (3) concentrating the cyclized peptide to 5-10 ml of solution, repurifying the peptide by reverse phase-high pressure liquid chromatography (RP-HPLC) and finally lyophilizing the peptide. In a specific embodiment, in which the peptide does not contain two appropriately situated cysteine residues, cysteine residues can be introduced at the amino-terminus and/or carboxy-terminus and/or internally such that the peptide to be cyclized contains two cysteine residues spaced such that the residues can form a disulfide bridge. Alternatively, a cyclic peptide can be obtained by generating an amide linkage using, for example but not limited to, the following protocol: An allyl protected amino acid, such as aspartate, glutamate, asparagine or glutamine, can be incorporated into the peptide as the first amino acid, and then the remaining amino acids are coupled on. The allyl protective group can be removed by a two hour mixing of the peptide-resin with a solution of tetrakistriphenylphophine palladium (0) in a solution of chloroform containing 5% acetic acid and 2.5% N-methylmorpholine. The peptide resin can be washed three times with 0.5% N,N-diisopropylethylamine (DIEA) and 0.5% sodium diethyldithiocabamate in DMF. The amino terminal Fmoc group on the peptide chain can be removed by two incubations for 15 minutes each in 20% piperdine in DMF, and washed three times with DMF for 30 seconds each. The activator mix, N-methylmorpholine and HBTU in DMF, can be brought onto the column and allowed to couple the free amino terminal end to the carboxyl group generated by removal of the allyl group to cyclize the peptide. The peptide can cleaved from the resin as described in the general description of chemical peptide synthesis above and the peptide purified by reverse phase-high pressure liquid chromatography (RP-HPLC). In a specific embodiment, in which the peptide to be cyclized does not contain an allyl protected amino acid, an allyl protected amino acid can be introduced into the sequence of the peptide, at the amino-terminus, carboxy-terminus or internally, such that the peptide can be cyclized.
xcex2-hCG peptides can also be obtained by recombinant expression techniques. (See, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 2d Ed., Cold Spring Harbor, N.Y., Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K., Vol. I, II). The nucleic acid sequence encoding hCG has been cloned and the sequence determined (see FIG. 8 and Xia, H., 1993, J. Molecular Endocrinology Jun. 10, 1993:337-343; Sherman, G. B., 1992, J. Molecular Endocrinology, Jun. 6, 1992:951-959; Gieseman, L. K. (ed.), 1991, Basic and Chemical Endocrinology, pp. 543-567; Ward et al., 1991, in Reproduction in Domestic Animals, 4th ed., P. T. Coppos, ed., pp. 25-80, Academic Press, New York) and can be isolated using well-known techniques in the art, such as screening a library, chemical synthesis, or polymerase chain reaction (PCR).
To recombinantly produce a xcex2-hCG peptide, a nucleic acid sequence encoding the xcex2-hCG peptide is operatively linked to a promoter such that the xcex2-hCG peptide is produced from said sequence. For example, a vector can be introduced into a cell, within which cell the vector or a portion thereof is expressed, producing the xcex2-hCG peptide. In a preferred embodiment, the nucleic acid is DNA if the source of RNA polymerase is DNA-directed RNA polymerase, but the nucleic acid may also be RNA if the source of polymerase is RNA-directed RNA polymerase or if reverse transcriptase is present in the cell or provided to produce DNA from the RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in bacterial or mammalian cells.
Expression of the sequence encoding the xcex2-hCG peptide can be by any promoter known in the art to act in bacterial or mammalian cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3xe2x80x2 long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the HSV-1 (herpes simplex virus-1) thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42), etc., as well as the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene control region which is active in erythroid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46, 89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropin releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378). The promoter element which is operatively linked to the nucleic acid encoding the xcex2-hCG peptide can also be a bacteriophage promoter with the source of the bacteriophage RNA polymerase expressed from a gene for the RNA polymerase on a separate plasmid, e.g., under the control of an inducible promoter, for example, a nucleic acid encoding the xcex2-hCG peptide operatively linked to the T7 RNA polymerase promoter with a separate plasmid encoding the T7 RNA polymerase.
In a less preferred embodiment, peptides can be obtained by proteolysis of hCG followed by purification using standard techniques such as chromatography (e.g., HPLC), electrophoresis, etc.
Also included within the scope of the invention are xcex2-hCG peptide derivatives which are differentially modified during or after synthesis, e.g., by benzylation, glycosylation, acetylation, phosphorylation, amidation, pegylation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. In specific embodiments, the peptides are acetylated at the N-terminus and/or amidated at the C-terminus. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
In another embodiment, the xcex2-hCG peptide derivative is a chimeric, or fusion, protein comprising a functional xcex2-hCG peptide joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein. In one embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a xcex2-hCG-coding sequence joined in-frame to a coding sequence for a different protein). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
Gene Therapy
In a specific embodiment, nucleic acids comprising a sequence encoding xcex2-hCG or a xcex2-hCG peptide or fused xcex2-hCG peptides (i.e., two or more xcex2-hCG peptides linked at the N-termini and C-termini via peptide bond(s)), are administered for treatment or prevention of HIV infection, wasting, and/or cancer, and/or promotion of hematopoiesis by way of gene therapy. Gene therapy refers to therapy performed by the administration of a nucleic acid to a subject. In this embodiment of the invention, the nucleic acid produces its encoded protein that mediates a therapeutic effect. For example, any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215. Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley and Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.
In a preferred aspect, the nucleic acid encoding xcex2-hCG or a xcex2-hCG peptide or fused xcex2-hCG peptides or related fusion protein is part of an expression vector that produces xcex2-hCG protein or a xcex2-hCG peptide or fused xcex2-hCG peptides or related fusion protein in a suitable host. In particular, such a nucleic acid has a promoter operably linked to the nucleic acid sequence coding for xcex2-hCG or a xcex2-hCG peptide or fused xcex2-hCG peptides or related fusion protein, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, a nucleic acid molecule is used in which the xcex2-hCG sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of xcex2-hCG (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then administered to the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering it in linkage to a peptide which is known to enter the cell or nucleus, e.g., by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to target cell types specifically expressing the receptors), etc. In a specific embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO92/06180 dated Apr. 16, 1992 (Wu et al.); WO92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.), WO93/20221 dated Oct. 14, 1993 (Young)). In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
In a specific embodiment, a viral vector that contains the nucleic acid sequence encoding xcex2-hCG or a xcex2-hCG peptide or fused xcex2-hCG peptides or related fusion protein is used. For example, a retroviral vector can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome. Retroviral vectors are maintained in infected cells by integration into genomic sites upon cell division. The nucleic acid to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.
Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; and Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234.
Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300.) Herpes viruses are other viruses that can also be used.
Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
The resulting recombinant cells can be delivered to a patient by various methods known in the art. In a preferred embodiment, recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are administered intravenously. Additionally, epithelial cells can be injected, e.g., subcutaneously, or recombinant skin cells (e.g., keratinocytes) may be applied as a skin graft onto the patient. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
In an embodiment in which recombinant cells are used in gene therapy, a nucleic acid sequence coding for xcex2-hCG or a xcex2-hCG peptide or fused xcex2-hCG peptides or related fusion protein is introduced into the cells such that it is expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells, preferably hematopoietic stem or progenitor cells, are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention.
Administration of hematopoietic, preferably hematopoietic stem and progenitor, cells which have been induced to proliferate with a Therapeutic of the invention and have undergone gene therapy, i.e., which have stably incorporated a heterologous gene capable of expression by their progeny cells, can be of great value in the treatment of diseases and disorders affecting cells of hematopoietic lineage. In one embodiment, hematopoietic reconstitution with such recombinant hematopoietic cells can be used in the treatment of genetic disorders of the hematopoietic system. Such genetic disorders include but are not limited to those listed in Table 1, supra. Genetic deficiencies or dysfunctions of hematopoietic cells can be treated by supplying, to a patient, recombinant stem and progenitor cells. In a specific embodiment, patients who have hematopoietic cells which lack a gene or have a mutant gene, can be provided stem and progenitor cells that have incorporated a functional counterpart of the deficient gene. In particular, such genes which can be subject to gene therapy include but are not limited to hemoglobin or enzymes which mediate its synthetic pathway (e.g., for treatment of anemias such as beta-thalassemia, sickle-cell disease).
In another specific embodiment, patients with infections by pathogenic microorganisms which occur in or affect a hematopoietic cell lineage can be treated with recombinant hematopoietic cells. Such recombinant hematopoietic cells can contain a heterologous gene which is expressed as a product which ameliorates disease symptoms, is toxic to the pathogen without significant detriment to the host, or interferes with the pathogen""s life cycle, etc. Pathogens which cause infections which may be treated with recombinant stem cells according to this embodiment of the invention include but are not limited to lymphotropic viruses such as HIV; gram-negative bacilli such as Brucella or Listeria; the mycobacterium which cause tuberculosis, or which cause Hansen""s disease (leprosy); parasites such as Plasmodium (the etiological agents of malaria), or Leishmania; and fungi (such as those that cause pneumonia and other lethal infections secondary to immunodeficiencies) (for a discussion of many of these disorders, see Harrison""s Principles of Internal Medicine, 1970, 6th Edition, Wintrobe, M. M., et al., eds., McGraw-Hill, New York, pp. 798-1044).
As a particular embodiment, it is possible to construct recombinant stem or progenitor cells that express a sequence which is xe2x80x9canti-sensexe2x80x9d to the nucleic acid of a hematopoietic cell pathogen. Such a sequence, which is complementary to the pathogen""s RNA or DNA, can hybridize to and inactivate such RNA or DNA, inhibiting the function or expression of the nucleic acid and disrupting the pathogen""s life cycle. As a particular example, recombinant hematopoietic cells can be used in the treatment of AIDS. Recombinant stem and progenitor cells which express an anti-sense nucleic acid that is complementary to a critical region (e.g., the long-terminal repeat or polymerase sequence) of the HIV genome (Wain-Hobson et al., 1985, Cell 40:9-17) can be used for hematopoietic reconstitution for the treatment of AIDS.
Many methods of gene therapy are available in the art (for general reviews of the methods of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley and Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.
In a preferred aspect, the nucleic acid which provides a gene product desired in a subject is introduced into an expression vector that produces the gene product. In particular, such a nucleic acid has a promoter operably linked to the nucleic acid sequence of interest, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, a nucleic acid molecule is used in which the sequences of interest are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the desired protein (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijistra et al., 1989, Nature 342:435-438).
In an embodiment of the invention, the nucleic acid is introduced into a hematopoietic cell that is then expanded by exposure to a Therapeutic of the invention prior to administration in vivo of the resulting recombinant cell. Alternatively, the nucleic acid can be introduced after expansion. Such introduction can be carried out by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be used to construct the recombinant hematopoietic cells for purposes of gene therapy. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny. The resulting recombinant cells can be delivered to a patient by various methods known in the art. In a preferred embodiment, recombinant hematopoietic cells are administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
Preparation of Hematopoietic Cells in vitro
Sources of hematopoietic stem and progenitor cells, which cells can be induced to proliferate according to one embodiment of the present invention, include but are not limited to bone marrow, fetal and neonatal blood (preferably from the umbilical cord and/or placenta), fetal liver, adult peripheral blood, neonatal thymus, and neonatal spleen. The foregoing list of sources is deemed to include cell samples (e.g., cryopreserved cells, cell lines, long-term cell cultures) derived therefrom. The source is mammalian, e.g., mouse, cow, horse, primate, monkey, and is most preferably human. Techniques for obtaining such stem and progenitor cells are well known in the art. For example, in one particular embodiment, human bone marrow cells can be obtained from the posterior iliac crest by needle aspiration (see, e.g., Kodo et al., 1984, J. Clin. Invest. 73:1377-1384). Neonatal blood can be obtained at birth by direct drainage from the umbilical cord and/or by needle aspiration from the delivered placenta at the root and at distended veins (see U.S. Pat. Nos. 5,004,681 and 5,192,553). Fetal blood can be obtained, e.g., by taking it from the fetal circulation at the placental root with the use of a needle guided by ultrasound (Daffos et al., 1985, Am. J. Obstet. Cynecol. 153:655-660; Daffos et al., 1983, Am. J. Obstet. Gynecol. 146:985), by placentocentesis (Valenti, 1973, Am. J. Obstet. Gynecol. 115:851; Cao et al., 1982, J. Med. Genet. 19:81), by fetoscopy (Rodeck, C. H., 1984, in Prenatal Diagnosis, Rodeck, C. H. and Nicolaides, K. H., eds., Royal College of Obstetricians and Gynaecologists, London), etc.
The method of the invention which comprises contacting hematopoietic stem and/or progenitor cells (or other hematopoietic cells) with a Therapeutic of the invention, can be carried out on unseparated, partially separated, or purified cell populations, before and/or after cryopreservation (and thawing) or in vitro culturing of such cell populations, before and/or after introduction of a recombinant gene, and any other desired manipulations of the cells. In a preferred aspect, samples (e.g. bone marrow or adult blood or neonatal blood) can be subjected to physical and/or immunological cell separation procedures so as to enrich for hematopoietic stem and progenitor cells (e.g., prior to culturing in the presence of a Therapeutic of the invention to induce proliferation of the cells).
Various procedures are known in the art and can be used to enrich for stem and progenitor cells. These include but are not limited to equilibrium density centrifugation, velocity sedimentation at unit gravity, immune rosetting and immune adherence, counterflow centrifugal elutriation, T lymphocyte depletion, and fluorescence-activated cell sorting, alone or in combination. Procedures have been reported for the isolation of very highly enriched populations of stem/progenitor cells. U.S. Pat. No. 5,061,620 dated Oct. 29, 1991 discloses a method for isolation of human hematopoietic stem cells. Murine CFU-S have been purified by several groups using slightly different procedures (Visser et al., 1984, J. Exp. Med. 59:1576; Nijhof et al., 1984, Exp. Cell Res. 155:583; Bauman et al., 1986, J. Cell. Physiol. 128:133; Lord and Spooncer, 1986, Lymphokine Res. 5:59). Studies using human (Emerson et al., 1985, J. Clin. Invest. 76:1286) or murine (Nicola et al., 1981, Blood 58:376) fetal liver cells have yielded highly enriched progenitor cells with up to 90% of them being colony forming cells for multi-, erythroid-, and granulocyte-macrophage lineages. CFU-E have also been very highly enriched (Nijhof et al., 1983, J. Cell Biol. 96:386). Purification of adult mouse marrow CFU-GM with cloning efficiencies of up to 99% in semi-solid medium has been accomplished by pretreatment of mice three days prior to sacrifice with cyclophosphamide, density separation of cells on Ficoll-Hypaque, and counterflow centrifugal elutriation (Williams et al., 1987, Exp. Hematol. 15:243). The resulting fraction of cells contained no detectable CFU-GEMM, BFU-E or CFU-MK, but up to 10% of the cells formed CFU-S measured at day 12. These procedures, or modifications thereof, can be used.
Human stem and progenitor cells are present in the non-adherent, low density, T-lymphocyte-depleted fraction of bone marrow, spleen, and adult and cord blood cells. Low density (density less than 1.077 gm/cm3) cells can be separated by use of Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, N.J.) or Percol (Broxmeyer, H. E., 1982, J. Clin. Invest. 69:632-642). In this procedure, the mature cells of the granulocytic series, which are not needed for transplantation, are removed in the dense fraction which goes to the bottom of the tube. An adherence/nonadherence separation protocol can also be used for enrichment of hematopoietic stem and progenitor cells.
It is also possible to use cell separation procedures that entail immunological recognition of cells. Stem and progenitor cells can be isolated by positive or negative selection using antibodies that recognize antigenic determinants on the surface of cells. One means is to separate the cells by using monoclonal antibodies which recognize cell surface determinants on these cells, in conjunction with separation procedures such as fluorescence-activated cell sorting or panning (Broxmeyer et al., 1984, J. Clin. Invest. 73:939-953). Human hematopoietic stem and progenitor cells contain antigenic determinants that are not present on all other cells, which can be used in antibody selection protocols for enrichment purposes; such antigens include but are not limited to those described infra.
Within the human system, several antigens have been found on stem/progenitor cells. The first antigenic system studied intensively was that of the MHC class II antigens, especially HLA-DR. This antigen has been found on CFU-GEMM, BFU-E, and CFU-GM (Lu et al., 1983, Blood 61:250; Winchester et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:4012; Busch et al., 1987, Blut 54:179). Several investigators have suggested that HLA-DR are not found, or are present at a low density on cells earlier than CFU-GEMM (Moore et al., 1980, Blood 55:682; Keating et al., 1984, Blood 64:1159).
Groups of antibodies have been used to distinguish different progenitors of the granulocyte-macrophage lineage (Ferrero et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4114). Type 1 CFU-GM contribute all of the peripheral blood CFU-GM, as well as a small number of bone marrow CFU-GM. They express surface antigens recognized by S3-13 and S17-25 antibodies, but not by R1B19 and WGHS-29-1 antibodies. Type 2 CFU-GM are present only in the marrow and react with S3-13, R1B19, and WGHS-29-1. Culture of type 1 CFU-GM in liquid culture generates type 2 CFU-GM. These antibodies have also been used to characterize CFU-GM from patients with chronic myeloproliferative disorders (Robak et al., 1985, Leukemia Res. 9:1023; Ferrero et al., 1986, Cancer Res. 46:975).
Other antigens on human stem/progenitor cells include those reactive with the My10 (Leary et al., 1987, Blood 69:953; Strauss et al., 1986, Exp. Hematol. 14:879), 3C5 (Katz et al., 1985, Leukemia Res. 9:191; Katz et al., 1986, Leukemia Res. 10:961), RFB-1 (Bodger et al., 1983, Blood 61:1006), 12-8 (Andrews et al., 1986, Blood 67:842), and L4F3 (Andrews et al., 1986, Blood 68:1030) antibodies. The antigen recognized by L4F3 is on CFU-GM, CFU-MK, BFU-E, and CFU-GEMM, but is apparently absent from cells which generate these progenitors in suspension culture (id.). The antigen recognized by the My10 antibody is CD34 (Civin et al., U.S. Pat. No. 4,714,680 dated Dec. 22, 1987.) Two subsets of pluripotent hematopoietic stem cells have been reported, a CD34+ HLA-DR+ CD38xe2x88x92 subset and a more primitive CD34+ HLA-DRxe2x88x92 CD38xe2x88x92 subset, with a gradual increase in CD38 expression as the hematopoietic cells proceed toward a more differentiated state (Huang and Terstappen, 1992, Nature 360:745-749; Terstappen et al., 1992, Leukemia 6:993-1000). The antigen recognized by another antibody, Myll, is expressed on CFU-GM, but not on BFU-E or CFU-GEMM (Strauss et al., 1986, Exp. Hematol. 14:935). Receptors for various lectins are also expressed on stem/progenitor cells (Nicola et al., 1980, J. Cell Physiol. 103:217; Reisner et al., 1982, Blood 59:360; Reisner et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:2933; Aizawa and Tavassoli, 1986, Int. J. Cell Cloning 4:464).
To expand the numbers of the hematopoietic stem and/or progenitor cells, the hematopoietic stem and/or progenitor cells (or precursor cells thereof) are exposed to or contacted with a composition comprising a Therapeutic of the invention for a sufficient time period, i.e., until the desired number of cells is obtained and the time period should be for as long as it is desired to keep cells self-renewing. Preferably, the cells are contacted with the Therapeutic, for example but not limited to, 200 IU/ml hCG (e.g., hCG APL) or xcex2-hCG preparation or a fraction of a source of hCG or xcex2-hCG or 100 xcexcg/ml xcex2-hCG peptide, preferably a xcex2-hCG peptide having the amino acid sequence of amino acid numbers 45-57 or 109-119 (SEQ ID NOS:6 or 7, respectively), or circularized peptide of amino acid numbers 44-57 (SEQ ID NO:12) with cysteine substituted for valine at position 44, or a branched peptide of amino acid numbers 45-57 (SEQ ID NO:6) with diaminobutyric acid substituted for the amino acids at positions 47 and 51 with proline peptide bonded to the diaminobutyric acid residues, or a circularized branched peptide of amino acids 44-57 (SEQ ID NO:12) with cysteine substituted for valine at position 44 and with diaminobutyric acid substituted for the amino acids at positions 47 and 51 with proline peptide bonded to the diaminobutyric acid residues or a peptide having the sequence of amino acids 45-57 (SEQ ID NO:6) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 109-119 (SEQ ID NO:7) or linked at the N-terminus via a peptide bond to the C-terminus of amino acids 110-119 (SEQ ID NO:27); or an amino acid sequence of amino acids 47-57 (SEQ ID NO:28) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 108-119 (SEQ ID NO:29) or the cyclic, fused peptide, having a sequence of amino acids 110-119 (SEQ ID NO:27) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 45-57 (SEQ ID NO:6) and being circularized by a disulfide bond between the terminal cysteines at positions 110 and 57, where all amino acid numbers are of the xcex2-hCG sequence depicted in FIG. 8 (a portion of SEQ ID NO:2), while under appropriate culture conditions, for a time period in the range of 1-21 or, more preferably, 7-21 days.
The composition comprising the Therapeutic of the invention, to which the stem and progenitor cells are exposed according to the invention, optionally also contains other growth factors and/or cytokines or cell culture materials, including but not limited to erythropoietin (Epo), Steel factor (SLF), IL-1, IL-3, IL-4, IL-6, IL-11, G-CSF, GM-CSF, FBS, adult PB plasma, alone or in combination. Preferably, factors are present that cause proliferation or, less preferably, differentiation of cells that are CFU-GEMM or earlier cells, e.g., IL-3, GM-CSF.
Contacting of the stem and progenitor cells with the Therapeutic preferably occurs during cell culture and thus, the Therapeutic is preferably added to the cell culture medium being used to culture the hematopoietic stem and/or progenitor cells. Such culturing can be by any method known in the art, including, but not limited to, cells grown in culture dishes, test tubes, roller bottles, bioreactors (perfusion system machines wherein cells are grown on a surface with continual perfusion by medium; e.g., as sold by Aastrom Biosciences, Inc., Ann Arbor, Mich.), etc. Various protocols have been described for the growth in vitro of cord blood or bone marrow cells, and it is envisioned that such procedures, or modifications thereof, may be employed (see, e.g. Smith, S. and Broxmeyer, H. E., 1986, Br. J. Haematol. 63:29-34; Dexter et al., 1977, J. Cell. Physiol. 91:335; Witlock and Witte, 1982, Proc. Natl. Acad. Sci. U.S.A. 79:3608-3612). The cell culture medium is supplemented to contain an effective concentration of the Therapeutic, for example but not limited to, 200 I.U. hCG (APL) or xcex2-hCG preparation or 100 xcexcg/ml of a xcex2-hCG peptide or a fraction of a native hCG or native xcex2-hCG preparation.
Progeny cells of hematopoietic stem and progenitor cells of can be generated in vitro; the differentiated progeny cells thus generated can be therapeutically useful. For example, in one embodiment of this aspect of the invention, hematopoietic stem cells and/or CFU-GEMM progenitor cells, can be induced to differentiate into platelets. Such platelets can be used, for example, for infusion into a patient with thrombocytopenia, such as, but not limited to, the ITP associated with HIV infection. In another embodiment, granulocytes can be generated in vitro prior to infusion into a patient. One or more of the hematopoietic progeny cells can be generated in vitro, allowing for the in vitro production of blood components. In one embodiment, the generation of differentiated blood components is accompanied by expansion of the hematopoietic stem and progenitor cell pool, in order to allow for production of a greater quantity of differentiated cells. Various growth factors can be used to promote expansion and/or differentiation of hematopoietic stem and progenitor cells, such as cytokines (growth factors) including, but not limited to, G-CSF, CSF-1, IL-3, IL-5, tumor necrosis factor-xcex2, and xcex1-interferon. The blood components which are thus produced have uses which are not limited to therapeutic uses in vivo. For example, such progeny cells can be used in vitro, e.g., for the production and isolation of hematopoietic cell products such as growth factors, antibodies, etc.
A specific embodiment of the invention relates to a method of increasing the amount of hematopoietic cells, which method comprises contacting in vitro a non-terminally differentiated hematopoietic cell with a composition comprising an amount of a Therapeutic of the invention effective to increase proliferation of the cell, under conditions suitable and for a time period sufficient to increase the numbers of said hematopoietic cell. For example, hematopoietic cell numbers can be increased by contacting a non-terminally differentiated hematopoietic cell (e.g., a cell isolated from bone marrow or blood, adult or fetal or umbilical cord blood) with a composition comprising 200 IU/ml hCG (e.g., hCG APL) or xcex2-hCG preparation or a fraction of a source of hCG or xcex2-hCG or 100 xcexcg/ml xcex2-hCG peptide, preferably a xcex2-hCG peptide having the amino acid sequence of amino acid numbers 45-57 or 109-119 (SEQ ID NOS:6 or 7, respectively), or circularized peptide of amino acid numbers 44-57 (SEQ ID NO:12) with cysteine substituted for valine at position 44, or a branched peptide of amino acid numbers 45-57 (SEQ ID NO:6) with diaminobutyric acid substituted for the amino acids at positions 47 and 51 with proline peptide bonded to the diaminobutyric acid residues, or a circularized branched peptide of amino acids 44-57 (SEQ ID NO:12) with cysteine substituted for valine at position 44 and with diaminobutyric acid substituted for the amino acids at positions 47 and 51 with proline peptide bonded to the diaminobutyric acid residues or a peptide having the sequence of amino acids 45-57 (SEQ ID NO:6) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 109-119 (SEQ ID NO:7) or linked at the N-terminus via a peptide bond to the C-terminus of amino acids 110-119 (SEQ ID NO:27); or an amino acid sequence of amino acids 47-57 (SEQ ID NO:28) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 108-119 (SEQ ID NO:29) or the cyclic, fused peptide, having a sequence of amino acids 110-119 (SEQ ID NO:27) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 45-57 (SEQ ID NO:6) and being circularized by a disulfide bond between the terminal cysteines at positions 110 and 57, where all amino acid numbers are of the xcex2-hCG sequence depicted in FIG. 8 (a portion of SEQ ID NO:2), and culturing the cell for at least ten days.
Demonstration of Therapeutic Utility
The Therapeutics of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. Any in vitro or in vivo assay known in the art to measure HIV infection or production can be used to test the efficacy of a Therapeutic of the invention. By way of example, and not by way of limitation, one could use any of the in vitro or in vivo assays described herein.
HIV
In an embodiment of the invention, a method of screening a preparation comprising hCG, an hCG alpha or hCG beta chain, fragment or derivative of hCG or said alpha or beta chain, or a fraction of a source of hCG or xcex2-hCG for anti-HIV activity is provided, which assay comprises assaying said preparation or fraction for the ability to inhibit HIV replication or expression of HIV RNA or protein. In one specific embodiment, the hCG preparation or fraction is assayed by a method comprising measuring HIV-1 p24 antigen levels in cultured hematopoietic cells acutely infected with HIV-1, which cells have been contacted with the preparation or fraction; and comparing the measured HIV-1 p24 antigen levels in the cells which have been contacted with the hCG preparation or fraction with said levels in cells not so contacted with the preparation or fraction, wherein a lower level in said contacted cells indicates that the preparation or fraction has anti-HIV activity. In another specific embodiment, the hCG preparation or fraction is assayed by a method comprising measuring the activity of a reporter gene product expressed from a construct in which the HIV-1 LTR is operably linked to said reporter gene, wherein said construct is present in cells which have been contacted with the preparation or fraction; and comparing the measured expression of said reporter gene in the cells which have been contacted with the preparation or fraction with said levels in such cells not so contacted, wherein a lower level in said contacted cells indicates that the preparation or fraction has anti-HIV activity. In another specific embodiment, the hCG preparation or fraction is assayed by a method comprising measuring HIV-1 derived RNA transcripts or HIV-1 antigen levels in HIV-1 transgenic mice administered the preparation or fraction; and comparing the measured transcript or antigen levels in the mice which have been administered the preparation or fraction with said levels in mice not so administered, wherein a lower level in said administered mice indicates that the preparation or fraction has anti-HIV activity. In yet another specific embodiment, the hCG preparation or fraction is assayed by a method comprising measuring SIV p27 antigen levels in the peripheral blood mononuclear cells of SIV infected monkeys administered the preparation or fraction; and comparing the measured antigen levels in the monkeys which have been exposed to the preparation or fraction with said levels in monkeys not so administered, wherein a lower level in said administered monkeys indicates that the preparation or fraction has anti-HIV activity.
By way of example, to assay a Therapeutic in vitro, one can examine the effect of the Therapeutic on HIV replication in cultured cells. Briefly, cultured hematopoietic cells (e.g., primary PBMCs, isolated macrophages, isolated CD4+ T cells or cultured H9 human T cells) are acutely infected with HIV-1 using titers known in the art to acutely infect cells in vitro, such as 105 TCID50/ml. Then, appropriate amounts of the Therapeutic are added to the cell culture media. Cultures are assayed 3 and 10 days after infection for HIV-1 production by measuring levels of p24 antigen using a commercially available ELISA assay. Reduction in p24 antigen levels over levels observed in untreated controls indicates the Therapeutic is effective for treatment of HIV infection.
Additionally, assays for HIV-1 LTR driven transcription are useful for testing the efficacy of Therapeutics of the invention. Specifically, a reporter gene, i.e., a gene the protein or RNA product of which is readily detected, such as, but not limited to, the gene for chloramphenicol acetyltransferase (CAT), is cloned into a DNA plasmid construct such that the transcription of the reporter gene is driven by the HIV-1 LTR promoter. The resulting construct is then introduced by transfection, or any other method known in the art, into a cultured cell line, such as, but not limited to, the human CD4+ T cell line HUT 78. After exposure of the transformed cells to the Therapeutic, transcription from the HIV-1 LTR is determined by measurement of CAT activity using techniques which are routine in the art. Reduction in HIV-1 LTR driven transcription demonstrates utility of the Therapeutic for treatment and/or prevention of HIV infection.
Exemplary tests in animal models are described briefly as follows: First, a Therapeutic of the invention is administered to mice transgenic for HIV-1, e.g., mice which have integrated molecular clone pNL4-3 containing 7.4 kb of the HIV-1 proviral genome deleted in the gag and pol genes (Dickie, P., et al., 1991, Virology 185:109-119). Skin biopsies taken from the mice are tested for HIV-1 gene expression by RT-PCR (reverse transcription-polymerase chain reaction) or for HIV-1 antigen expression, such as expression of gp120 or NEF, by immunostaining. Additionally, the mice are examined for reduction in the cachexia and growth retardation usually observed in HIV-1 transgenic mice (Franks, R. R., et al., 1995, Pediatric Res. 37:56-63).
The efficacy of Therapeutics of the invention can also be determined in SIV infected rhesus monkeys (see Letrin, N. L., and King, N. W., 1990, J. AIDS 3:1023-1040), particularly rhesus monkeys infected with SIVmac251, which SIV strain induces a syndrome in experimentally infected monkeys which is very similar to human AIDS (Kestler, H., et al., 1990, Science 248:1109-1112). Specifically, monkeys can be infected with cell free SiVmac251, for example, with virus at a titer of 104.5 TCID50/ml. Infection is monitored by the appearance of SIV p27 antigen in PBMCs. Utility of the Therapeutic is characterized by normal weight gain, decrease in SIV titer in PBMCs and an increase in CD4+ T cells.
Once the Therapeutic has been tested in vitro, and also preferably in a non-human animal model, the utility of the Therapeutic can be determined in human subjects. The efficacy of treatment with a Therapeutic can be assessed by measurement of various parameters of HIV infection and HIV associated disease. Specifically, the change in viral load can be determined by quantitative assays for plasma HIV-1 RNA using quantitative RT-PCR (Van Gemen, B., et al., 1994, J. Virol. Methods 49:157-168; Chen, Y. H., et al., 1992, AIDS 6:533-539) or by assays for viral production from isolated PBMCs. Viral production from PBMCs is determined by co-culturing PBMCs from the subject with H9 cells and subsequent measurement of HIV-1 titers using an ELISA assay for p24 antigen levels (Popovic, M., et al., 1984, Science 204:497-500). Another indicator of plasma HIV levels and AIDS progression is the production of inflammatory cytokines such as IL-6, IL-8 and TNF-xcex1; thus, efficacy of the Therapeutic can be assessed by ELISA tests for reduction of serum levels of any or all of these cytokines. Administration of the Therapeutic can also be evaluated by assessing changes in CD4+ T cell levels, body weight, or any other physical condition associated with HIV infection or AIDS or AIDS Related Complex (ARC). Reduction in HIV viral load or production, increase in CD4+ T cell or amelioration of HIV-associated symptoms demonstrates utility of a Therapeutic for administration in treatment/prevention of HIV infection.
Wasting
Specific embodiments provide a method for screening a preparation comprising hCG or an hCG xcex1 chain or hCG xcex2 chain or a derivative of hCG or of said alpha or beta chain or a fraction of native hCG or native xcex2-hCG, for anti-wasting activity comprising assaying said preparation for the ability to promote weight gain in an animal model that exhibits a wasting syndrome. In one specific embodiment, the hCG preparation is screened by a method comprising measuring the weight of an offspring of an HIV-1 transgenic mouse, which offspring has been exposed to the preparation; and comparing the weight of the offspring which has been exposed to the preparation with the weight of an offspring not so exposed, wherein a greater weight in said exposed offspring indicates that the preparation has anti-wasting activity. In another specific embodiment, the hCG preparation is screened by a method comprising measuring the change in weight of an SIV infected monkey which has been exposed to the preparation; and comparing the change in weight of the monkey which has been exposed to the preparation to the change in weight of a monkey which has not been so exposed, wherein a greater weight increase or smaller weight loss in said exposed monkey indicates that the preparation has anti-wasting activity.
Any animal model in which wasting occurs can be used. Exemplary tests in animal models are described briefly as follows: First, a Therapeutic of the invention can be assayed in mice transgenic for HIV-1, e.g., mice which have integrated molecular clone pNL4-3 containing 7.4 kb of the HIV-1 proviral genome deleted in the gag and pol genes (Dickie, P., et al., 1991, Virology 185:109-119). These mice exhibit cachexia and growth retardation (Franks, R. R., et al., 1995, Pediatric Res. 37:56-63). A Therapeutic which reverses the cachexia and growth retardation in the HIV transgenic mice is predicted to have utility for treatment or prevention of wasting syndromes.
Similarly, the efficacy of Therapeutics of the invention can also be assayed in SIV infected rhesus monkeys (see Letrin, N. L., and King, N. W., 1990, J. AIDS 3:1023-1040), particularly rhesus monkeys infected with SIVmac251, which SIV strain induces a syndrome in experimentally infected monkeys which is very similar to human AIDS and results in weight loss in the infected monkeys (Kestler, H., et al., 1990, Science 248:1109-1112). Specifically, monkeys are infected with cell free SIVmac251, for example, with virus at a titer of 104.5 TCID50/ml and SIV infection is monitored by the appearance of SIV p27 antigen in PBMCs. An increase in the weight of infected monkeys indicates that the Therapeutic has utility in the treatment of wasting syndrome.
Compounds for use in therapy can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, prior to administration to humans, any animal model system known in the art may be used.
Once the Therapeutic has been tested in a non-human animal model, the utility of the Therapeutic can be determined in human subjects. Improvement in wasting syndrome, i.e. and increase in body cell mass, can be assessed by any well known clinical techniques available in the art. For example but not limited to, measuring body weight, determination of total body potassium content, determination of intracellular water volume, bioelectrical impedance analysis, anthropometrics and determination of total body nitrogen content (see, e.g., Kotler, D. P. et al., 1985, Am. J. Clin. Nutr. 42:1255-65; Ott, M. et al., 1993, Am. J. Clin. Nutr. 57:15-19; Miller, T. L. et al., 1993, Am. J. Clin. Nutr. 57:588-592). Therapeutics, the administration of which increases body weight or cell mass, should have utility in treatment of wasting syndrome.
Cancer
One embodiment provides a method for screening a preparation comprising a protein, preferably a purified protein, having a sequence of one or more portions of xcex2-hCG or a derivative, preferably in purified form, of said protein, or a fraction of a source of native hCG or native xcex2-hCG for anti-cancer activity comprising assaying said preparation for the ability to inhibit the survival or proliferation of malignant cells. In a specific embodiment, the preparation is screened by a method comprising measuring the survival or proliferation of malignant cells, which cells have been contacted with the preparation; and comparing the survival or proliferation of the cells contacted with the preparation with the survival or proliferation of cells not so contacted with the preparation, wherein a lower level of survival or proliferation in said contacted cells indicates that the preparation has anti-cancer activity. In another specific embodiment, the preparation is screened by a method comprising measuring the survival or proliferation of cells from a cell line which is derived from or displays characteristics associated with a malignant disorder, which cells have been contacted with the preparation; and comparing the survival or proliferation in the cells which have been contacted with the preparation with said survival or proliferation in cells not so contacted, wherein a lower level in said contacted cells indicates that the preparation has anti-tumor activity.
Another embodiment provides a method for screening a preparation comprising a protein having a sequence of a portion of xcex2-hCG or a derivative of said protein or a fraction of a source of native hCG or native xcex2-hCG, for anti-cancer activity comprising assaying said preparation for the ability to convert cells having an abnormal phenotype to a more normal cell phenotype. In a specific embodiment, the preparation is screened by a method comprising assessing the phenotype of cells suspected of being pre-neoplastic in culture, which cells have been contacted with the preparation; and comparing the phenotype in the cells which have been contacted with the preparation with said phenotype in cells not so contacted, wherein a more normal phenotype in said contacted cells indicates that the preparation has anti-cancer activity. In another specific embodiment, the preparation is screened by a method comprising assessing the phenotype of cells from a cell line which is derived from or displays characteristics associated with a pre-malignant disorder, which cells have been contacted with the preparation; and comparing the phenotype in the cells which have been contacted with the preparation with said phenotype in cells not so contacted, wherein a more normal phenotype in said contacted cells indicates that the preparation has anti-cancer activity.
Yet another embodiment provides a method for screening a preparation comprising a protein having a sequence of a portion of xcex2-hCG or a derivative of said protein or a fraction of a source of native hCG or native xcex2-hCG, for activity in treatment or prevention of Kaposi""s Sarcoma comprising assaying said preparation for the ability to inhibit Kaposi""s Sarcoma cell proliferation or promote Kaposi""s Sarcoma cell apoptosis. In a specific embodiment, the preparation is screened by a method comprising measuring proliferation or colony formation in cultured KS Y-1 or KS-SLK cells, which cells have been contacted with the preparation; and comparing the measured proliferation or colony formation in the cells which have been contacted with the preparation with said proliferation or colony formation in cells not so contacted with the preparation, wherein a lower level of proliferation or colony formation in said contacted cells indicates that the preparation has anti-Kaposi""s Sarcoma activity.
In another specific embodiment, the preparation is screened by a method comprising measuring apoptosis in a Kaposi""s Sarcoma tumor in an immunodeficient mouse, which Kaposi""s Sarcoma tumors have been induced by injection with KS Y-1 or KS-SLK cells, and which mouse has been exposed to the preparation; and comparing the degree of apoptosis in the tumor of the mouse which has been exposed to the preparation with a tumor in a mouse not so exposed, wherein a higher in level of apoptosis in the tumor of said exposed mouse indicates that the preparation has anti-Kaposi""s Sarcoma activity.
For example, in vitro assays which can be used to determine whether administration of a specific Therapeutic is indicated include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a Therapeutic, and the effect of such Therapeutic upon the tissue sample is observed. In one embodiment, where the patient has a malignancy, a sample of cells from such malignancy is plated out or grown in culture, and the cells are then exposed to a Therapeutic. A Therapeutic which inhibits survival or growth of the malignant cells is selected for therapeutic use in vivo. Many assays standard in the art can be used to assess such survival and/or growth; for example, cell proliferation can be assayed by measuring 3H-thymidine incorporation, by direct cell count, by detecting changes in transcriptional activity of known genes such as proto-oncogenes (e.g., fos, myc) or cell cycle markers; cell viability can be assessed by trypan blue staining, differentiation can be assessed visually based on changes in morphology, etc.
In various specific embodiments, in vitro assays can be carried out with representative cells of cell types involved in a patient""s disorder, to determine if a Therapeutic has a desired effect upon such cell types.
In another embodiment, cells of a patient tissue sample suspected of being pre-neoplastic are similarly plated out or grown in vitro, and exposed to a Therapeutic. The Therapeutic which results in a cell phenotype that is more normal (i.e., less representative of a pre-neoplastic state, neoplastic state, malignant state, or transformed phenotype) is selected for therapeutic use. Many assays standard in the art can be used to assess whether a pre-neoplastic state, neoplastic state, or a transformed or malignant phenotype, is present. For example, characteristics associated with a transformed phenotype (a set of in vitro characteristics associated with a tumorigenic ability in vivo) include a more rounded cell morphology, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, release of proteases such as plasminogen activator, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton surface protein, etc. (see Luria et al., 1978, General Virology, 3d Ed., John Wiley and Sons, New York pp. 436-446).
In other specific embodiments, the in vitro assays described supra can be carried out using a cell line, rather than a cell sample derived from the specific patient to be treated, in which the cell line is derived from or displays characteristic(s) associated with the malignant, neoplastic or pre-neoplastic disorder desired to be treated or prevented, or is derived from the cell type upon which an effect is desired, according to the present invention.
Specifically, Therapeutics can be tested for efficacy in treatment or prevention of Kaposi""s sarcoma by any of the methods relating to Kaposi""s sarcoma described herein or in Lunardi-Iskandar et al. (1995, Nature 375:64-68) or by any other method known in the art. Briefly, KS cell lines, KS Y-1 (Ibid.) or KS-SLK (Siegal, B. et al., 1990, Cancer 65:492-498), which will produce malignant tumors in immunodeficient mice, are used to perform in vitro proliferation and clonogenic assays (see, e.g., Lunardi-Iskandar, Y. et al., 1993, J. Exp. Med. 177:741-750); methods for performing such assays are well known in the art. A Therapeutic which reduces proliferation or colony formation in the cultured cells can be used in the methods of the invention for treatment or prevention of KS.
Efficacy of a Therapeutic can also be determined by administration of the Therapeutic to immunodeficient mice injected with either the KS-Y-1 or KS-SLK cells, which cause tumor formation in the mice, and assessment of the degree of apoptosis and angiogenesis of tumor cells after treatment with the Therapeutic. Apoptosis is detected by staining fixed tissue samples from the tumor for the presence of cells with DNA fragmentation. For example, this is accomplished by treating tissue slides from formalin-fixed tumors with terminal deoxynucleotide transferase for extension of DNA ends (3xe2x80x2 hydroxyl ends) and incorporation of digoxigenin-11-dUTP. Anti-digoxigenin antibody conjugated with the enzyme peroxidase allows detection of apoptotic cells that stain brown whereas viable cells stain blue. An increase in KS tumor cell apoptosis and a decrease in angiogenesis indicates that the Therapeutic has utility in treatment of KS.
The Therapeutic can also be assessed in clinical trials in human patients suffering from KS or any other cancer. To test the efficacy of the Therapeutic in KS patients, either local, i.e. intralesional, or systemic administration of the Therapeutic can be used. Tumors can be examined physically for regression in response to administration of the Therapeutic. Additionally, tissue biopsies can be taken from the tumors, and these tissue samples examined for apoptosis, as described above.
Compounds for use in therapy can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, prior to administration to humans, any animal model system known in the art may be used.
Hematopoiesis
The Therapeutics of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. Any in vitro or in vivo assay known in the art to measure a pro-hematopoietic effect, i.e. the ability to induce hematopoietic cell proliferation in vitro or production of one or more hematopoietic cell types in vivo, such as those described herein, can be used to test the efficacy of a Therapeutic of the invention.
A specific embodiment provides a method for screening a preparation comprising hCG or an hCG xcex1 chain or hCG xcex2 chain or a derivative of hCG or of said alpha or beta chain or a fraction of a source of native hCG or native xcex2-hCG, for pro-hematopoietic activity comprising assaying said preparation for the ability to induce an increase in hematopoietic cell numbers. In one embodiment, the preparation is screened by a method comprising measuring the number of colonies formed from hematopoietic stem or progenitor cells, which cells have been contacted with the preparation; and comparing the number of colonies formed from the cells contacted with the preparation with the number of colonies formed from cells not so contacted with the preparation, wherein a higher number of colonies formed from said contacted cells indicates that the preparation has pro-hematopoietic activity. In another embodiment, the preparation is screened by a method comprising measuring the number of CD4+ T cells in an SIV infected monkey, which monkey has been exposed to the preparation; and comparing the number of CD4+ T cells in the monkey which has been exposed to the preparation with the number of CD4+ T cells in a monkey not so exposed, wherein a higher number of CD4+ T cells in said exposed monkey indicates that the preparation has pro-hematopoietic activity.
Specifically, to assay a Therapeutic in vitro, one could examine the effect of the Therapeutic on proliferation of hematopoietic cells in vitro. For example, to assay colony-forming units (a progenitor cell), briefly, the hematopoietic cells are cultured for an appropriate amount of time, such as 5 to 20 days and preferably 10 days, in the presence of (or otherwise exposed to) the Therapeutic to be tested, and then colony assays are performed to determine the number of colonies formed in comparison to the number of colonies formed by cells cultured in the absence of the Therapeutic. For example, hematopoietic progenitor cells can be isolated from bone marrow or cord blood, seeded in methylcellulose in the presence of absence of the Therapeutic, and then colony numbers determined after 10 days of culture. An increase in colony numbers in cells contacted with the Therapeutic indicates that the Therapeutic has activity in inducing proliferation of hematopoietic cells. Thus, for example, depending on the progenitor cell desired to be assayed, CFU-GM, CFU-GEMM, etc., assays can be done.
Therapeutics can also be tested in vivo for activity in increasing the numbers of hematopoietic cells. Preferably, Therapeutics are tested in animal models of hematopoietic disorders before testing them in human patients. For example, but not by way of limitation, a Therapeutic can be tested in rhesus monkeys infected with SIV, particularly SIVmac251 which induces a syndrome in monkeys similar to human AIDS (Kestler, H. et al., 1990, Science 248:1109-1112), and which are deficient in CD4+ T cells. The Therapeutic to be tested can be administered to the infected monkeys; then the blood or bone marrow of the infected monkeys can be examined for an increase in CD4+ T cells or any other hematopoietic cell type for which the monkey is deficient. An increase in numbers of the hematopoietic cell demonstrates that the Therapeutic is useful for treating diseases and disorders associated with hematopoietic deficiencies. Any animal model of an anemia can be similarly used for testing.
Therapeutics can be tested in human patients, preferably after tests in vitro and/or in vivo in an animal model, with hematopoietic deficiencies, for example but not limited to, deficiencies associated with HIV infection such as anemia, neutropenia, thrombocytopenia, or CD4+ T cell lymphocyte deficiency, for activity in increasing numbers of hematopoietic cells for which the patient is deficient. Briefly, the Therapeutic is administered, for example by intramuscular injection two to three times per week, to the patient suffering from the hematopoietic deficiency. The subject""s blood or bone marrow is assayed before and after treatment with the Therapeutic for an increase in the hematopoietic cell numbers. Therapeutics which cause an increase in hematopoietic cell numbers are useful for treatment of diseases and disorders associated with hematopoietic deficiencies.
Assays for hematopoietic cell proliferation in the blood or bone marrow can be accomplished by any method well known in the art. For example, blood can be drawn and blood cell numbers can be determined by routine clinical laboratory tests for red blood cells, platelets, neutrophils, lymphocytes, etc. Additionally, colony assays on isolated bone marrow can be performed to assess increases in stem or progenitor cells. For example, bone marrow can be sampled and bone marrow cells evaluated for stem and progenitor cell colony formation. Briefly, cells are seeded in methylcellulose, cultured for 12 to 14 days, and then scored for colony formation where aggregates containing more than 50 cells are counted as a colony (see, e.g., Lunardi-Iskandar, Y. et al., 1995, Nature 375:64-68; Louache, R. et al., 1992, Blood 180:2991-2999; Lunardi-Iskandar, Y. et al., 1989, J. Clin. Invest. 83:610-615). Bone marrow progenitors which can be evaluated by this colony assay include, but are not limited to, CFU-Mix, BFU-e and CFU-GM. As an alternative to colony assays for detection and quantitation of stem and/or progenitor cells, immunological detection methods can be employed, based on the antigens expressed by the particular cell type (see, e.g., the relevant discussion hereinabove).
Therapeutic Compositions and Methods of Administration
The invention provides methods of treatment and prevention by administration to a subject in need of such treatment of a therapeutically or prophylactically effective amount of a Therapeutic of the invention. The subject is preferably an animal, including, but not limited to, animals such as monkeys, cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human. In a specific embodiment, the subject is a human not afflicted with a cancer which secretes hCG or hCG fragments and, more particularly, not afflicted with KS.
Various delivery systems are known and can be used to administer a Therapeutic of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the Therapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a Therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved, for example and not by way of limitation, by topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
In another embodiment, the Therapeutic can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the Therapeutic can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
In a specific embodiment where the Therapeutic is a nucleic acid encoding a protein Therapeutic, the nucleic acid can be administered by gene therapy methods as described herein.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier. In a specific embodiment, the term xe2x80x9cpharmaceutically acceptablexe2x80x9d means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
The term xe2x80x9ccarrierxe2x80x9d refers to a diluent, adjuvant, excipient, or vehicle with which the Therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in xe2x80x9cRemington""s Pharmaceutical Sciencesxe2x80x9d by E. W. Martin. Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The Therapeutics of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The amount of the Therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vivo and/or in vitro assays may optionally be employed to help predict optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient""s circumstances. Doses of, for example but not limited to, at least 15,000 I.U. and up to 45,000 I.U. hCG weekly was effective and well tolerated in humans. Weekly doses of 6,000 I.U. in monkeys and 300-500 I.U. in mice were also effective. Predicted suitable doses of a hCG peptide for treatment or prevention of HIV infection include, but are not limited to, 1 to 1000 micrograms per week. Routes of administration of a Therapeutic include, but are not limited to, intramuscularly, subcutaneously or intravenously. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.